Methods of determining an analyte concentration, as well as devices and test elements incorporating the same

ABSTRACT

Methods, devices and test elements are disclosed for detecting at least one analyte in a sample. The methods, devices and test elements are used for detecting one or more analytes such as metabolites in body fluids, especially glucose. The methods, devices and test elements correct for an interfering variable such as temperature and or hematocrit in a test element system via a correction factor based upon diffusion of a diffusable label.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of Int'l Patent ApplicationNo. PCT/EP2012/076361; filed 20 Dec. 2012, which claims priority to andthe benefit of EP Patent Application No. 11195083.8; filed 22 Dec. 2011.Each patent application is incorporated herein by reference as if setforth in its entirety.

TECHNICAL FIELD

This disclosure relates generally to chemistry and medical diagnostics,and more particularly, it relates to methods of correctingtemperature-dependence and/or hematocrit-dependence of a test elementsystem when determining an analyte concentration via a correction factorbased upon diffusion of a diffusable label.

BACKGROUND

In many areas of the natural sciences and technology, it is necessary toreliably and rapidly detect in a liquid and/or gaseous sample one ormore analytes in a qualitative and/or quantitative manner. For example,as part of preventive diabetes care and/or diabetes treatment, it isgenerally necessary to determine blood sugar level at least once a day,generally multiple times, to guide individuals having diabetes to takeappropriate countermeasures if deviations from a normal value or rangeoccur.

So that the daily routine of such individuals is not compromised anymore than is necessary, devices and methods have been developed thatallow blood sugar measurements not only in a clinical environment butalso in the workplace or home, as well as during leisure activities.Such devices and methods generally are based on using one or moredisposable test elements, which are known and available in differentforms. For example, test elements can be in the form of test strips,test tapes, test disks, test needles or in other forms.

Test elements often include one or more test fields having at least oneanalyte-specific detection reagent as part of the test chemistry. Thedetection reagent is selected and designed for carrying out a detectablereaction in the presence of the analyte of interest. Examples ofcommercially available test elements include, but are not limited to,Accu-Chek® Aviva, Accu-Chek® Performa, Accu-Chek® Active, Accu-Chek® Goor Accu-Chek® Mobile test cassette with appropriate test instrumentssuch as Accu-Chek® Aviva, Active, Go and Mobile from Roche DiagnosticsOperations, Inc. (Indianapolis, Ind.)

Int'l Patent Application Publication No. WO 2010/052306 describes adiagnostic test element for detecting an analyte of interest in a bodyfluid sample. The test element includes a test field having a detectionreagent configured to undergo a detectable optical change in thepresence of the analyte. The test field has at least one detection layerthat includes the detection reagent and that has particles with at least90% of all the particles of the detection layer having an actualparticle size of less than 10 μm.

Likewise, Int'l Patent Application Publication No. WO 2010/052307describes a test element for detecting an analyte of interest in asample. The test element includes at least one test field having a testfield surface and having a detection reagent configured to carry out adetectable reaction in the presence of the analyte. In addition, thetest element has at least one distribution element having at least onedistribution surface facing the test field surface with at least onecapillary gap being formed between the distribution surface and the testfield surface. In test elements, it is possible to use one or moredifferent test chemistries, such as an enzyme system, to convert anddetect the analyte.

To provide a user with a manageable form of the enzyme system, theenzyme system can be introduced or immobilized on the test element in adry and solid layer(s) as at least part of the test chemistry togetherwith further reactive substances such as a mediator. The user only hasto apply the sample to the layer to obtain a measurement result shortlythereafter. As such, after the sample is applied to the layer of thetest element, the analyte-specific detection reaction proceeds both inthe layer and completely or partly in the sample. For example, one ormore reactive substances in the form of the detection reagent orconstituents thereof can dissolve in the sample and can be observed upto an end point of the reaction.

These methods also can involve diffusion processes, in which the sampletransports the analyte to the reactive substances and/or the detectionsubstance formed diffuses into or out of a detection layer. For example,the end point of the reaction can be determined in electrochemical oroptical systems. In this manner, the rate of formation of a detectionsubstance can be equal to the rate of diffusion of the detectionsubstance formed. Alternatively, a measurement signal, from which theanalyte concentration can be derived, is determined at a particular timeafter applying the sample to the test field.

Such diffusion processes, as well as enzyme-catalyzed reactions, may betemperature-dependent. In addition, further constituents of the sample,such as cellular and/or particulate constituents (e.g., red blood cells)may have an influence on the diffusion and dissolution processes in thetest element. The result can be a heavy dependence of the measuredanalyte concentration on the ambient temperature at which themeasurement is carried out, as well as on the hematocrit (Hct) of thesample applied to the test element. Thus, in principle, there might bethe risk of analyte concentration being influenced, and this in turn canlead to a challenge when dosing medicaments such as insulin. Especiallyin low concentration ranges of analytes, such as blood glucose in thecase of a diabetic, an incorrectly high measured blood glucose can leadto too much insulin being administered. Therefore, it is important tocorrect the temperature-dependence and/or Hct-dependence of theparticular test element system.

For example, temperature can be measured by a sensor accommodated in themeasurement instrument and used to correct analyte concentration.However, this can in turn lead to faulty corrections, since temperaturegenerally is not measured at the actual position of the chemicalreaction of the analyte. Consequently, the temperature used forcorrection can differ from the temperature of the reaction site of theanalyte, and so the correction can in turn lead to errors.

Furthermore, it is difficult to carry out a Hct correction, especiallyin optical systems since there are generally no purely Hct-dependentmeasurement values that could be used to correct analyte concentration.With electrochemical systems, however, correcting the measurement signalwith regard to Hct is possible but is very laborious owing to a complexpulse sequence method and is additionally superimposed by otherinterfering signals in the sample.

In this manner, U.S. Pat. No. 4,250,257 describes methods and devicesfor analyzing whole blood samples. The methods and devices use a gel inwhich an inert substance is accommodated and diffuses out of the gelinto the whole blood sample, whereas plasma of the whole blood samplediffuses into the gel. The diffusion of the inert substance out of thegel is inversely proportional to the Hct of the blood sample. Inaddition, various possibilities of bringing about a Hct correction aredisclosed. In one example, a separate gel can be used to determine Hct,and from this a correction factor subsequently can be used for anotherblood sample. Alternatively, an analyte detection reaction and a Hctcorrection can be carried out by means of two different color changes,with a first color change resulting from the analyte detection reactionand with a second color change resulting from diffusion of a dye asinert medium into the sample. Furthermore, a Hct correction usingalbumin is described with bromocresol green (BCG).

Additionally, U.S. Pat. No. 7,548,773 describes a method of calibratinga measurement system based upon dissolution of an analyte in a referencechannel. Owing to the dissolution of a known amount of analyte by samplein the reference channel, it is possible to carry out, in parallel tothe actual analyte concentration determination, a differentialdetermination relating to the reference channel to determine thereaction rate of the sample. As a result, corrections can be made on theanalyte concentration. However, a disadvantage of this method is theneed for dissolving a reference molecule to be determined on a capillarywall. The method also can be influenced by properties of the system thatare not associated with the sample. Furthermore, the dissolution andmovement of the analyte-identical calibrator is dependent on the analyteconcentration. It is not possible to distinguish between influences ofanalyte concentration and other factors on the movement of thecalibrator, and this can in turn lead to falsification of the determinedanalyte concentration.

For the foregoing reasons, there is a need for additional methods,devices and test elements that correct or compensate fortemperature-dependence and/or Hct-dependence of a test element systemwhen determining an analyte concentration.

BRIEF SUMMARY

This disclosure describes devices, test elements and methods ofdetecting an analyte in a body fluid sample that correct or compensatean analyte concentration for interfering variables that may be presentin the body fluid sample, devices and test elements used therein. Toaddress the disadvantages noted above, an inventive concept providedherein is that such interfering variables can be corrected orcompensated by detecting diffusion of a diffusable label andincorporating the detected diffusion into correction information thatalso takes into account an influence of the interfering variable ondiffusion of the diffusable label.

It is therefore an object of the present disclosure to provide devices,test elements and methods of detecting at least one analyte in a samplethat at least partly avoid the disadvantages of known methods, devicesand test elements as discussed above. More particularly, the disclosureprovides methods, devices and test elements that at least partly avoidfalsification of a measured analyte concentration owing to diffusioneffects, even in the case of varying Hct and temperature of the sampleor of the test element. A further object is to provide methods, devicesand test elements for detecting at least one analyte in a sample thatallow analyte-independent detection.

In one aspect, devices are provided that include at least one analytedetector. In addition, the devices include at least one diffusable labeldetector. In some instances, the devices also include an evaluation unitconfigured to completely or partly carry out the methods describedherein.

In another aspect, test elements are provided that include a testchemistry situated in a detection layer of a test field mounted on atest support of the test element. The test chemistry can be configuredfor electrochemically or optically detecting the analyte.

In some instances, the test chemistry is a detection reagent thatincludes at least one enzymatic detection reagent. The detection reagentalso can include a mixture of multiple detection reagents or multiplesubstances that together form the detection reagent that may convert theanalyte to a detectable signal. Examples of analyte-specific enzymaticdetection reagents include, but are not limited to, oxioreductaseenzymes (e.g., GlucDor/PQQ), dehydrogenase enzymes, oxidase enzymes orsimilar enzymes or combinations thereof. In other instances, the atleast one enzymatic detection reagent is glucose oxidase (GOD) orglucose dehydrogenase (e.g., FAD-, NAD+- or PQQ-dependent GDH).

The test chemistry can be in the form of at least one test chemistrylayer or at least one detection layer. The at least one test chemistrylayer optionally can include substances such as, for example, one ormore fillers. In some instances, the filler can be one or more types ofparticles such as inorganic particles, which are not identical to thedetection reagent or at least not completely identical to the detectionreagent. The test chemistry layer also can include at least one organicfilm former.

The test elements also include at least one diffusable label. In someinstances, the diffusable label can be a dye and/or can include at leastone such dye, the diffusion behavior of which can be ascertained byelectrochemical or optical detection methods in the test element, thesample and/or at least part of the sample. In other instances, thediffusable label is a cyanine dye, azo dye, sulfone dye, or acombination of at least two thereof. In still other instances, thediffusable label can be erioglaucine, indigo carmine, hydroxynaphtholblue, 1,1-diethyl-4,4-carbocyanine iodide, amaranth, or a combination ofat least two thereof.

The test elements can be designed in a wide variety of configurations.In some instances, the test elements are test strips, test tapes, testneedles and/or microsamplers.

In view of the foregoing, methods are provided for determining at leastone analyte concentration in a body fluid sample such as blood orinterstitial fluid. Alternatively, the method can be used for detectingat least one metabolite in a body fluid sample.

The methods can include a step of performing a calibration measurementto obtain at least one piece of correction information based upondiffusion of a diffusable label, where the at least one piece ofcorrection information accounts for an influence of at least oneinterfering variable in a body fluid sample and accounts for a generalrelationship between the at least one interfering variable and diffusionof the diffusable label.

The methods also include a step of detecting diffusion of at least onediffusable label in a body fluid sample or at least part of the bodyfluid sample having or suspected of having an analyte of interesttherein that has been applied to at least one test element as describedherein. The at least one piece of correction information is generatedfrom the diffusion of the diffusable label.

The methods also include a step of detecting or measuring an analyteconcentration in the body fluid sample with the at least one testelement as described herein. Thus, at least one reaction is detectedbetween the test element's test chemistry and the analyte of interest.

The detecting steps can be an electrochemical or optical detectablereaction. However, other types of detection reactions are contemplated.

In some instances, the reaction between the test chemistry and theanalyte can be one in which at least one detection substance is formedin the presence of the at least one analyte. In this manner, it is alsopossible to form and/or use multiple detection substances which can bedetected individually, in groups or altogether. Detection substances arein particular substances that are formed owing to the at least onedetection reaction and/or that are involved in the at least onedetection reaction and which are directly or indirectly detectable. Onthe basis of the at least one detection substance detected, it ispossible, for example, for the at least one analyte to be detectedquantitatively and/or qualitatively.

Regardless of whether the detecting step is an electrochemical oroptical detectable reaction, at least one signal, which is also referredto as a measurement value, is generated. The at least one signal can beascertained during and/or after reaction of the test chemistry with theanalyte and can be corrected by the at least one piece of correctioninformation. In some instances, the at least one piece of correctioninformation is about the actual temperature and/or the Hct.

The methods also include the step of correcting or compensating theanalyte concentration by taking into account the at least one piece ofcorrection information, where the at least one piece of correctioninformation corrects or compensates for at least one interferingvariable of the sample. In some instances, the piece of correctioninformation takes into account an influence of at least one of thefollowing interfering variables during the determination of theconcentration of the analyte: a temperature of the sample and/or of thetest element, a proportion of constituents of at least one substance inthe sample such as Hct.

These and other advantages, effects, features and objects of theinventive concept will become better understood from the descriptionthat follows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the inventive concept.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages, effects, features and objects other than those set forthabove will become more readily apparent when consideration is given tothe detailed description below. Such detailed description makesreference to the following drawings, wherein:

FIG. 1 a shows a diagram of an exemplary test element having a testfield.

FIG. 1 b shows a diagram of an exemplary test field containing adiffusable label before wetting of the test field.

FIG. 1 c shows a diagram of an exemplary test field containing adiffusable label after wetting of the test field.

FIG. 1 d shows a diagram of an exemplary device having an exemplary testelement.

FIG. 1 e shows a diagram of an exemplary device having an alternativetest element.

FIG. 1 f shows a modification of the exemplary embodiment according toFIG. 1 e.

FIG. 2 shows curve profiles after wetting of a test field including0.05% erioglaucine at different temperatures with samples of varyingglucose content.

FIG. 3 shows curve profiles after wetting of a test field including 0.1%erioglaucine at different temperatures.

FIG. 4 shows curve profiles after wetting of a test field includinghydroxynaphthol blue at different temperatures.

FIG. 5 shows curve profiles after wetting of a test field including0.05% erioglaucine with blood or water at different temperatures.

FIG. 6 shows curve profiles after wetting of a test field including0.05% erioglaucine with blood containing varying Hct.

FIG. 7 shows curve profiles of measurements on different test fieldsincluding 0.05% erioglaucine with samples having varying Hct at varyingglucose concentrations.

FIG. 8 shows curve profiles of test fields including erioglaucine withsamples having varying Hct at varying glucose concentrations.

FIG. 9 shows curve profiles of test fields including 0.05% or 0.1%erioglaucine.

FIG. 10 shows a diagram of the influence of temperature and Hct on ananalyte measurement signal after 4 seconds.

FIG. 11 shows a diagram of the influence of temperature and Hct on thereflectance behavior of a diffusable label.

FIG. 12 shows a correlation between a system error during a glucosemeasurement and a reflectance measurement on a diffusable label field atvarying Hct.

FIG. 13 shows a graph of system error of a conventional test elementsystem at varying Hct without using a piece of correction informationfrom the diffusable label.

FIG. 14 shows a graph of system error of an exemplary test elementsystem at varying Hct using a piece of correction information from thediffusable label

While the inventive concept is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments thatfollows is not intended to limit the inventive concept to the particularforms disclosed, but on the contrary, the intention is to cover alladvantages, effects, features and objects falling within the spirit andscope thereof as defined by the embodiments described herein and theclaims below. Reference should therefore be made to the embodimentsdescribed herein and claims below for interpreting the scope of theinventive concept. As such, it should be noted that the embodimentsdescribed herein may have advantages, effects, features and objectsuseful in solving other problems.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The devices, test elements and methods now will be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the inventive concept are shown. Indeed, thedevices, test elements and methods may be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein; rather, these embodiments are provided so that thisdisclosure will satisfy applicable legal requirements.

Likewise, many modifications and other embodiments of the devices, testelements and methods described herein will come to mind to one of skillin the art to which the disclosure pertains having the benefit of theteachings presented in the foregoing descriptions and the associateddrawings. Therefore, it is to be understood that the devices, testelements and methods are not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims. Although specificterms are employed herein, they are used in a generic and descriptivesense only and not for purposes of limitation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the disclosure pertains. Although any methods and materialssimilar to or equivalent to those described herein can be used in thepractice or testing of the devices, test elements and methods, thepreferred methods and materials are described herein.

Moreover, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one element is present,unless the context clearly requires that there be one and only oneelement. The indefinite article “a” or “an” thus usually means “at leastone.” Likewise, the terms “have,” “comprise” or “include” or anyarbitrary grammatical variations thereof are used in a non-exclusiveway. Thus, these terms may both refer to a situation in which, besidesthe feature introduced by these terms, no further features are presentin the entity described in this context and to a situation in which oneor more further features are present. For example, the expressions “Ahas B,” “A comprises B” and “A includes B” may both refer to a situationin which, besides B, no other element is present in A (i.e., a situationin which A solely and exclusively consists of B) or to a situation inwhich, besides B, one or more further elements are present in A, such aselement C, elements C and D, or even further elements.

Overview

Exemplary devices, test elements and methods are provided and are basedupon the inventive concept of using a diffusable label to obtain atleast one piece of correction information to correct for an interferingvariable on an analyte concentration.

As used herein, “piece of correction information” means at least onepiece of information about at least one interfering variable, it beingpossible to derive the piece of information from diffusion of adiffusable label and the interfering variable having, or it beingpossible for the interfering variable, an influence on determininganalyte concentration.

As used herein, “interfering variable” means at least one property orcombination of properties of a sample, which property or combination isindependent of the actual analyte concentration, and which can influencea detection of a reaction of the test chemistry with the analyte and/orthe actual reaction of the test chemistry with the analyte and caninfluence and/or falsify determining analyte concentration. Likewise,the at least one interfering variable can describe a proportion of atleast one interfering component in the sample (i.e., a proportion of atleast one substance of the sample), which influences the reaction of thetest chemistry with the analyte and/or which influences the detection ofthe reaction of the test chemistry with the analyte. For example, oneinterfering variable can be a Hct value of the sample. Alternatively oradditionally, the interfering variable can be a temperature of thesample and/or of the environment. Alternatively still, the interferingvariable can be Hct and temperature of the sample and/or of theenvironment. Generally, a reaction of the analyte with the testchemistry is slowed down by lowering the temperature of the sampleand/or of the environment. Conversely, the reaction of the analyte isgenerally increased when Hct in the sample is lowered.

The devices, test elements and methods are useful in a variety ofapplications. For example, the devices, test elements and methods areuseful as diagnostic tools for a variety of analytes. Examples ofanalytes of interest include, but are not limited to cholesterol,lactate, fructose and glucose. The description below, however, islargely directed toward glucose, as it is generally the most importantanalyte when monitoring blood of individuals having diabetes. It is tobe understood that the inventive concept is transferrable to otheranalytes of interest.

Devices

Devices of the inventive concept include at least one analyte detector,which can be at least one electrochemical analyte detector and/or atleast one optical analyte detector. These types of detectors are knownto one of skill in the art.

With respect to electrochemical analyte detectors, they includeelectrodes that contact the reagent to be analyzed. Electrochemicalanalyte detectors have at least one, or even two or more, electrodes forelectrochemical detection.

With respect to optical analyte detectors, they include at least onephotosensitive detector element, such as at least one photodiode formedcompletely or partly differently from an optional photosensitivedetector element of the diffusable label detector as described below.Alternatively or additionally, the photosensitive detector element ofthe optical analyte detector can be formed completely or partlyidentically in terms of components to the photosensitive detectorelement of the diffusable label detector. For example, a commonphotodiode can be provided that is both a component of the opticalanalyte detector and a component of the diffusable label detector. Ifthe diffusable label detector and the optical analyte detector share atleast one component, it is possible to carry out a temporally separatedor staggered detection of the diffusable label and/or of the diffusionof the diffusable label and of the analyte by using a pulsed and/orintermittent measurement scheme in which the diffusion of label isacquired at particular times by means of the photodiode and the analytedetection is carried out at other times by means of the same photodiode.The optical analyte detector also can include at least one light sourcesuch as, for example, at least one light-emitting diode (LED),configured to illuminate at least one test field containing the testchemistry. The at least one light source of the optical analyte detectorcan be designed completely or partly differently from the at least onelight source of the diffusable label detector.

As part of the inventive concept, the devices also include at least onediffusable label detector, which can be at least one electrochemicaldiffusable label detector and/or at least one optical diffusable labeldetector. As above, these types of detectors are known to one of skillin the art.

With respect to the electrochemical diffusable label detector, it caninclude one or more electrochemical detection electrodes.

With respect to the optical diffusable label detector, it can include atleast one photosensitive detector element such as at least onephotodiode to detect light emitted by a light source. The light sourcecan be at least one LED, it being possible to configure the light sourcefor illuminating at least one test chemistry field containing thediffusable label and/or at least one label field containing the labeland/or for illuminating the sample.

In this manner, the light of the light source need not be directeddirectly onto the detector, but instead can be altered or reflected byvarious elements, such as, for example, filters, mirrors or the testelement.

The light source for the optical detectors can radiate light within anydesired wavelength range from about 200 nm and about 1000 nm. In someinstances, the light source can radiate light at about 200 nm, about 300nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800nm, about 900 nm or about 1000 nm. Depending on the wavelength rangewithin which the analyte absorbs the light, another wavelength range maybe used for detecting the diffusable label. For example, light within aUV light wavelength range from about 200 nm to about 400 nm can be usedfor exciting the diffusable label when the analyte absorbs light withina wavelength range from about 500 nm to about 1000 nm. Conversely, lightwithin a wavelength rage from about 300 nm to about 400 nm can be usedfor exciting the analyte when the diffusable label absorbs light withina wavelength range from about 400 nm to about 1000 nm. Other wavelengthranges for the excitation of the analyte or the diffusable label arelikewise conceivable.

As used herein, “about” means within a statistically meaningful range ofa value or values such as a stated concentration, length, molecularweight, pH, time frame, temperature, volume or wavelength. Such a valueor range can be within an order of magnitude, typically within 20%, moretypically within 10%, and even more typically within 5% of a given valueor range. The allowable variation encompassed by “about” will dependupon the particular system under study, and can be readily appreciatedby one of skill in the art.

The devices also can include at least one evaluation unit in the form ofa data processing device. The evaluation unit can be configured tocompletely or partly carry out the methods as described herein.

Test Elements

Test elements of the inventive concept include a test field having atleast one test chemistry. As used herein, “test field” means an elementthat has a cohesive quantity of test chemistry such as, for example, atleast one layer of the test chemistry and also optionally one or morefurther components.

The at least one test chemistry is configured to carry out at least onedetectable reaction with the analyte. As used herein, “test chemistry”means a substance and/or a mixture configured to carry out at least onedetectable change of at least one property in the presence of theanalyte of interest to be detected. More particularly, the testchemistry can contain at least one reagent that converts the analyte ina detection reaction. Int'l Patent Application Publication No. WO2010/052307 discloses an exemplary test chemistry and detection reagentthat may be used in the test elements described herein.

As used herein, “detectable reaction” means a reaction that isdetectable by means of at least one physical (i.e., optical) and/orelectrochemical detection method. Detectable reactions are known thatare based upon detecting the analyte of interest by optical and/orelectrochemical means. In this manner, it is possible to use reactionsin which at least one detection substance is formed that can be detectedoptically and/or electrochemically.

One or more detection reagents for detecting the analyte in the samplecan be situated in the test chemistry.

The test elements can, in particular, have a form as may be known in theart such as, for example, a test strip, a test tape, a test needle or amicrosampler (i.e., an element having at least one needle or lancet andat least one capillary element). However, other forms for the testelements are also possible in principle.

The test elements thus include at least one test field, which can be atwo- or three-dimensional region of the test element usable fordetecting the analyte. In some instances, the test field is a dry testfield. The test field includes the test chemistry having at least onedetection reagent configured to carry out a detectable reaction in thepresence of the analyte. In addition, the test field can include furthersubstances such as, for example, carriers, auxiliaries, pigments,fillers, buffer substances and the like. In some instances, thedetection reagent can include at least one enzymatic detection reagent.

The test elements therefore include at least one test field having atleast one test chemistry layer, where the test chemistry layer includesthe test chemistry such as at least one enzyme. Example of enzymes foruse in the test fields include, but are not limited to, glucosedehydrogenase and glucose oxidase.

As noted above, the at least one test chemistry can be in the form of atest chemistry layer. The test chemistry layer can, for example, bedesigned analogously to the first film layer of the test element asdescribed in EP Patent No. 0 821 234.

The test chemistry layer also can include at least one organic filmformer such as, for example, a polyvinyl proprionate dispersion.Alternatively or additionally, other film formers may be used.

As part of the inventive concept, the test element also includes atleast one diffusable label, where the at least one diffusable label issituated in at least one first region of the test element, and where thediffusable label is configured to diffuse at least partly from the firstregion of the test element into at least one second region As usedherein, “diffusable label” means a detectable chemical substance that isat least locally determinable by electrochemical and/or opticaldetection methods.

The at least one diffusable label is chemically different from the atleast one analyte of interest. As used herein, “chemically different”mean that the individual molecule of the label deviates by at least oneatom from the analyte of interest. For example, the molecule of thelabel can have at least one atom more or less than the analyte ofinterest. Alternatively, “chemically different” means that the numberand sequence of atoms in the diffusable label are identical to thesequence of atoms in the analyte of interest, but the spatialarrangement of atoms is not identical at every position, as occurs, forexample, in the case of compounds having asymmetric carbon atoms (i.e.,two enantiomers of a same molecule). For instance, one possibility wouldbe to use two enantiomers, one of the enantiomers representing theanalyte and the other enantiomer representing the diffusable label.

As used herein “diffusable label different from the analyte of interest”means a label that is at least partly non-identical to the analyte ofinterest. For example, by the label having at least one chemicalsubstance that is not in the analyte of interest that is to be detected.

As used herein, “locally” means a defined area or a defined volume ofthe test field used for detecting the diffusable label. For example, andwith respect to optically detecting the diffusable label, the area orthe volume can be defined by the form of the incident light or by thepositioning of the detector. With respect to electrochemically detectingthe label, it is possible for the area or the volume to be defined bythe form and arrangement of the electrode.

As noted above, the at least one diffusable label can be situated in oneregion of the test field or in a further region of the test element,where the at least one diffusable label is configured to diffuse atleast partly from the at least first region of the test element into atleast one second region. The second region can be a region within thetest field or outside the test field, or within the test element oroutside the test element.

One region for this purpose is the supernatant of the sample above thetest element. For example, the diffusable label can diffuse from a firstregion of the test element into a second region that is a supernatant ofthe sample. The diffusable label can be situated together with the testchemistry in a test chemistry layer and can diffuse into the sampleduring wetting of the test field of the test element with the sample,into at least one second region that can be either within or outside thetest element.

Alternatively or additionally, the diffusable label also can at least becompletely or partly arranged outside the test element before contactingof the test element and/or the test chemistry. The label can be designedsuch that upon contacting the test element with the sample, at least onedetectable diffusion of the label occurs in the sample or at least oneconstituent of the sample. The diffusion can take place in a supernatantof the sample that has not been taken up by the test element and/or thetest chemistry such as, for example, a drop-shaped supernatant, and/orin part of the sample that has been taken up by the test element or apart thereof.

As such, the diffusable label can be contained in at least one region ofthe test elements at least before wetting with sample, where the testchemistry is likewise completely or partly contained in the firstregion. The region can be referred to as a first region. If thediffusable label is present only in this at least one first region ofthe test element, the region also can be referred to as a label region.The first region therefore can be designed as a label layer.

In this manner, the test chemistry layer and the diffusable label layercan be identical. Once the sample contacts the diffusable label and thetest chemistry, it is possible for both diffusable label and testchemistry in the test element and/or in the sample to diffuse into oneor more regions having a lower concentration. Owing to differences inconcentration in different regions of the test field, or of the sample,of diffusable label and test chemistry, it is possible for bothdiffusable label and test chemistry to diffuse into regions of thesample having a lower concentration of these constituents. It isadvantageous when diffusion behavior, more particularly diffusion rate,of the diffusable label resembles the diffusion behavior or thediffusion rate of the test chemistry and the analyte. In this manner, itis possible for both the diffusable label and the analyte to be detectedin a same region of the test field.

When the diffusable label and the test chemistry are completely orpartly situated in a first region of the test element, detectingdiffusion of the label can be achieved with the same detector and/orfrom the same side of the test element. As noted above, this can becarried out both electrochemically and optically. However, it ispossible to use at least one distinct detector for both the diffusablelabel and the test chemistry (i.e., at least one diffusable labeldetector and at least one analyte detector). For detecting diffusion ofthe diffusable label, a region of the test element that includes thelabel before wetting of the test element with sample can be measured, oralternatively, a region of the test element or the sample that does notinclude any diffusable label before wetting of the label with sample canbe measured. In the former, a decrease in label concentration followingwetting of the label with sample is consequently measured, whereas inthe latter, the increase in concentration of the label in the measuredregion takes place.

Briefly, the diffusable label can be or can include a substance that isintroduced into the test element in addition to the test chemistry. Thediffusable label does not react and/or interact with the test chemistryand/or constituents of the test chemistry. This ensures that thediffusion behavior of the diffusable label is substantially independentof the behavior of the test chemistry and/or of the analyte and ensuresan independent way of determining properties of the sample to obtain apiece of correction information, thereby increasing accuracy of ananalyte concentration determination.

The diffusion behavior of the dye can be ascertained by electrochemicalor optical detection methods in the test element, the sample and/or atleast part of the sample.

In some instances, the label can include at least one opticallydetectable substance such as, for example, at least one dye (e.g., atleast one inorganic or organic dye). As used herein, “dye” means asubstance (i.e., a chemical compound) that can carry out at least onedetectable interaction with light in the ultraviolet and/or visibleand/or infrared spectral range such as, for example, reflection withchange of a wavelength and/or with change of spectral properties of thereflected light, scattering with change of a wavelength and/or withchange of spectral properties of the scattered light, fluorescence,phosphorescence or a combination thereof.

As used herein, “optically detectable dye” means a substance havingelectrons that interact with electromagnetic waves such as a wavelengthrange from about 100 nm to about 1500 nm (referred to hereinafter aslight), from about 100 nm to about 400 nm (also referred to asultraviolet light), from about 300 nm to about 800 nm (referred tohereinafter as visible light), and/or from about 800 nm to about 1500 nm(also referred to as infrared light). In other instances, the dyeinteracts with wavelengths of about 100 nm, about 200 nm, about 300 nm,about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm,about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1300nm, about 1400 nm or about 1500 nm. Interactions can be an absorption,fluorescence or phosphorescence. For example, the dye can be selected sothat it spectrally influences the electromagnetic waves in at least onewavelength range or at at least one wavelength by the dye selectivelyabsorbing light at one or more wavelengths and/or in at least onewavelength range. This spectral influence ought to be detectable.Alternatively or additionally, the wavelength of the light can bealtered so that there is a shift in wavelength from incident light toemitted and optionally detected light. As such, the dye can include anabsorption dye and/or a fluorescence dye and/or a phosphorescence dye.

Moreover, the dye can be an organic dye and/or an inorganic dye.Depending on the intended use, it may be advantageous for the dye tohave hydrophilic or hydrophobic properties. When using aqueous samplessuch as body fluids (e.g., blood, plasma, serum, urine or sputum), thedye should be at least to some extent water-soluble. Additionally, thediffusable label has good chemical stability and good light stabilityand availability.

Examples of dyes that can be used include, but are not limited to,cyanine dyes, azo dyes, sulfone dyes, or combinations of at least twothereof. Owing to their charge distribution within the dye molecule, thedyes are suitable for absorbing light within a particular wavelengthrange. As a result of the absorption of the light, their position can bemade determinable. To this end, light of a particular wavelength can bedirected into the region to be detected and, as a result of an increaseor decrease in intensity of the reflected light in the region inrelation to a preceding time, the presence of the diffusable label andthe label diffusing away is made detectable.

Possible cyanine dyes are all cyanine dyes known to one of skill in theart for detection purposes. Specific examples of cyanine dyes include,but are not limited to, streptocyanine or open-chain cyanine,hemicyanine and closed-chain cyanine such as phthalocyanine, formazan,porphyrins and 1,1-diethyl-4,4-carbocyanine iodide, or combinations ofat least two thereof.

Possible azo dyes are all azo dyes known to one of skill in the art fordetection purposes. Specific examples of azo dyes include, but are notlimited to, aliphatic and aromatic azo compounds such as aniline yellow,methyl orange, azobenzene, hydroxynaphthol blue,4-(dimethylaniline)azobenzene, amaranth, allura red, azorubine,anthocyanins, para red, or combinations of at least two thereof.

Possible sulfone dyes are all sulfone dyes known to one of skill in theart for detection purposes. Specific examples of sulfone dyes include,but are not limited to, aliphatic and aromatic sulfonic acids such asalkylbenzenesulfonic acids and alkylbenzenesulfonates, Brilliant BlueFCF (i.e., erioglaucine), azorubine and naphthalenesulfonic acid, orcombinations of at least two thereof.

In some instances, the diffusable label can be erioglaucine, indigocarmine, hydroxynaphthol blue, 1,1-diethyl-4,4-carbocyanine iodide oramaranth. Erioglaucine absorbs light of a wavelength of about 625 nm,indigo carmine absorbs light of a wavelength of about 608 nm,thydroxynaphthol blue absorbs light of a wavelength of about 650 nm,1,1-diethyl-4,4-carbocyanine iodide absorbs light of a wavelength ofabout 703 nm or about 648 nm, and amaranth absorbs light of a wavelengthof about 521 nm. In other instances, the diffusable label iserioglaucine, hydroxynaphthol blue, or a combination thereof.

These dyes generally have a diffusion behavior that is virtuallyindependent of the presence or the concentration of the analyte.Conversely, the diffusable label should influence the behavior of theanalyte as little as possible. However, it is also possible to use otherdyes that exhibit a dependence of their diffusion rate in a sample onthe temperature or on particulate or cellular constituents (e.g., Hct).A list of the structural formulae relating to the aforementioned dyesand the absorption maximum thereof can be found in Table 1 in theexperimental section.

The diffusable label can be further configured so that it interacts withthe analyte and/or with the test chemistry as little as possible or notat all, where a diffusion rate of the label is substantially independentof the concentration of the analyte. What ought to be achieved is thatthe detection reaction proceeds undisturbed by the diffusable label andthe diffusion of the label in each case. Thus, the diffusion rate of thediffusable label is substantially independent of the concentration ofthe analyte. For example, chemical reactions or other interactionsbetween, firstly, diffusable label, and, secondly, analyte or testchemistry are generally undesired. The interaction between, firstly,diffusable label and, secondly, analyte and test chemistry ought to beso low that the signals obtained using analyte and test chemistrywithout the diffusable label deviate by less than about 3%, by less thanabout 2% or by less than about 1% from signals obtained frommeasurements with analyte and test chemistry in the presence of thediffusable label.

To keep an interaction between the diffusable label and the analyte aslow as possible, the diffusable label can be situated in a region which,while coming into contact with the sample to be analyzed, does not comeinto contact with the test chemistry.

In some instances, the diffusable label can be arranged completely orpartly separately from the test chemistry. The label can be arrangedcompletely or partly separately from the test chemistry at least beforewetting of the test element and/or of the diffusable label with sample.In this manner, the diffusable label is prevented from being influencedby the test chemistry and the test chemistry is prevented from beinginfluenced by the diffusable label, at least during the wetting process.The separation of the diffusable label from the test chemistry can beachieved by a separate test field or by a separate test field layer withthe diffusable label but without test chemistry or detection reagentbeing arranged on the test element. Alternatively or additionally, it ispossible to use a distinct test element containing the diffusable label,which test element, while being wetted with the same sample at the sametime and under the same temperature conditions, does not contain anydetection reagents. Alternatively or additionally, an influence on thediffusion rate of the diffusable label by the analyte can be determinedexperimentally beforehand, and this can be taken into account in theconcentration calculation method described in more detail below. Inother instances, the diffusable label and the test chemistry arearranged in the same test field.

The test elements have two opposite surfaces. As such, the analytedetector and/or the diffusable label detector therefore can irradiatethe test elements or a part thereof from one side (i.e., a detectionside) with light. Reflected or scattered light emanating from the testelements can be detected from an opposite side from the detection sideby using the photosensitive detector element of the diffusable labeldetector and/or the photosensitive detector element of the analytedetector.

Such test elements can be wetted with the sample from the opposite side,which can also be referred to as a sample input side or application sideand which can face the detection side. For example, the test elementscan include at least one sample input/application side and at least onedetection side. Alternatively or additionally, the sample can be appliedat another site such as, for example, at a capillary element of the testelement.

The test elements also can have two spatially separate regions fordetecting the analyte and for detecting the diffusable label. Forexample, two adjacent regions of the test elements can be arranged inrelation to one another so that they both can be contacted with thesample but no fluid exchange is possible between them (i.e., nodiffusion is possible from one region to the other). Alternatively, theregions for detecting the analyte and for detecting the diffusable labelcan be arranged next to one another, and in each case diffusion of theanalyte and/or diffusion of the label shall be possible. As such, thesetwo regions can be arranged in relation to one another so that whenintroduced into a measurement instrument, they can be illuminated by alight source in each case. The region in which the diffusable label issituated alternatively can be formed as a capillary, which is filledupon wetting with the sample. For detecting the diffusable label, adiffusion of the label in the capillary can be determined.

The test elements also can include at least one partition layer thatretains at least one constituent of the sample such as, for example,particulate and/or cellular constituents including red blood cells,white blood cells and/or platelets. Alternatively or additionally, theat least one partition layer can include at least one optical materialsuch as, for example, at least one pigment for forming a reflectionlayer in the form of a pigment layer. Examples of optical materialinclude, but are not limited to, TiO₂, ZrO₂ or BaSO₄. In some instances,the partition layer is situated on the test field. More specifically,the partition layer can be situated on the side of the test element onwhich the sample is applied (i.e., the sample input side). For example,the sample can be applied on the side of the test element having thepartition layer, whereas the detection of the analyte can be achievedoptically on the opposite side. During penetration of the sample intothe test element, the sample or parts of the sample can penetrate one ormore layers of the test field such as the partition layer and come intocontact during the penetrating with both the test chemistry andoptionally the diffusable label. In addition, part of the sample canremain in or above the partition layer as a supernatant or part of asupernatant.

The test elements therefore can have at least one label layer and/or atleast one label field that includes the at least one diffusable label.As used herein, “layer” or “field” means a cohesive quantity of theparticular material, which quantity has a lateral extent and athickness. For example, the layer and/or the field can have a lateralextent such as a diameter and/or an equivalent diameter and/or a sidelength of at least about 100 μm, at least about 500 μm or at least about1 mm. In addition, the layer and/or the field can have a thickness ofless than about 100 μm or even less than about 50 μm. A layer can be acomponent of a multilayered assembly. The field can have an accessiblesurface that can be contacted with a liquid sample. Alternatively, thefield can be covered by one or more further layers and/or elements.

The test elements also can have one or more test fields that include theat least one test chemistry. Thus, the at least one label can becompletely or partly contained in the at least one test field or becompletely or partly mixed into the at least one test chemistry and/orbe contained in another way in the test field. In some instances, the atleast one label can be arranged completely or partly separately from thetest chemistry, but on the same test element and the same test support.As used herein, “separately” means that the test chemistry and the labelare not contained in a common, cohesive material, for example, not inthe same layer and/or not in the same layer assembly. The diffusablelabel can be arranged in particular in at least one label layer at leastbefore wetting with the sample, which label layer is arranged adjacentto or spaced apart from the test chemistry layer on adjacent orspaced-apart areas on a test support of the test element. The areas canbe spaced apart by at least about 100 μm, by at least about 200 μm or byat least about 500 μm. Alternatively or additionally, the at least onelabel layer can be arranged separated from the test chemistry layer byone or more layers in a layer assembly including at least one testchemistry layer and at least one label layer different from the testchemistry layer. In this manner, the label layer and the test chemistrylayer can directly adjoin to one another, or one or more further layerscan be arranged between the label layer and the test chemistry layer.Alternatively, the diffusable label can be arranged in at least onelabel layer above and/or below the test field. The label layer can be acomponent of the test element (e.g., the test field), but also can be alayer separate from the test element, which layer is nevertheless wettedby the same sample. Alternatively, although the label layer can bearranged on the test element, it is not a component of the test field.By separating the label from the test chemistry, it can be ensured thatthe label does not influence the diffusion properties and reactionproperties of the test chemistry. Again alternatively or additionally,the label and/or the label layer can be completely or partly arrangedoutside the test element, for example in a separate correction elementand/or in a device for determining at least one concentration of atleast one analyte in a sample, for example a test instrument.

Methods

Methods of the inventive concept include a step of performing acalibration measurement to obtain at least one piece of correctioninformation. Thus, at least one calibration measurement is acquired,which can be a general relationship between at least one interferingvariable and diffusion of the diffusable label. The general relationshipcan be reported in the form of one or more calibration curves. As usedherein, “general relationship” means a rule for a plurality of differentvalues of the interfering variable, which rule describes how the valuesof the interfering variable influence the diffusion of the diffusablelabel. The rule can be ascertained for a continuous range of values ofthe interfering variable or for a discontinuous range of values such as,for example, a quantity of interfering variable values spaced apart fromone another. Accordingly, the general relationship can include apointwise assignment of multiple interfering variable values to, in eachcase, a corresponding influence on label diffusion. Alternatively oradditionally, the rule can be a law in the form of an analyticalfunction, which can be referred to as a calibration curve or calibrationfunction and which describes analytically the influence on labeldiffusion by the interfering variable.

In some instances, the calibration measurement can be carried out bydetecting, in each case, at least one diffusion of at least onediffusable label in a plurality of test samples or calibration samplesin which the interfering variable is known. For example, it is possibleto prepare test samples having a known Hct and/or temperature. Withrespect to such test samples, it is possible in each case to ascertainat least one value of a diffusion of the diffusable label. As explainedin more detail below, it is possible to ascertain with respect to eachtest sample at least one optical measurement value (e.g., at least onereflectance value) of a label field in contact with the test sample. Inthis manner, it is possible to determine a quantity of pairs of values,which each include the interfering variable and the associated diffusionof diffusable label. The pairs of values can themselves describe thegeneral relationship, or the general relationship can be ascertainedfrom the pairs of values by means of a fit. In some instances, it ispossible for the general relationship to be described by a straightline, where the slope and axis intercept can be readily determined fromthe pairs of values by an appropriate fit. The straight line then can beused as a calibration curve. More complex calibration curves also arepossible such as, for example, exponential functions and/or polynomialsthat describe the relationship between the pairs of values.

The general relationship, more particularly the calibration curve orcalibration function, can be stored in at least one data storage devicesuch as, for example, a volatile and/or non-volatile data storage deviceof the devices described above such as, for example, an evaluation unitof the device as describe above.

As noted above, and as part of the inventive concept, the methods use atleast one diffusable label. As noted above, in some instances, diffusionof the diffusable label from at least one first region into at least onesecond region can be detected. The first and/or second region can eachbe regions of the test element. It is clear that there are amultiplicity of options for detecting diffusion by a change inconcentration being observed locally.

As such, diffusion of the at least one diffusable label in the sample orat least one constituent thereof can be caused by differences inconcentration of the diffusable label such as, for example, between thefirst and second region. As a result of wetting of the test elementand/or of the diffusable label, a difference in concentration can arisebetween at least one first region, in which the label is situated beforewetting, and at least one second region such as, for example, asupernatant of the sample and/or a region of the test element and/or ofa correction element different from the first region, and so the labeldiffuses owing to the difference in concentration.

The diffusion of the diffusable label can be detected electrochemicallyor optically. For example, one or more diffusable label detectors can beused that can be configured to detect the diffusion of the diffusablelabel.

Moreover, a concentration of the diffusable label in at least oneobservation volume in the first region and/or the second region can beacquired at at least two different times or at multiple times. Forexample, a particular volume in which the diffusable label is situatedbefore wetting with sample can be measured electrochemically and/oroptically. After wetting of the volume and other volumes of the testfield, the concentration of the diffusable label in the volume will sinkowing to the differences in label concentration. Alternatively oradditionally, it is possible to observe a particular volume that buildsup with the diffusable label after wetting with sample.

The diffusion rate of the diffusable label generally depends on manydifferent parameters, such as, for example, temperature, pressure andthe chemical environment of the diffusable label. As used herein,“chemical environment of the label” means the environment of the labelbefore wetting with sample and/or the environment of the diffusablelabel after wetting with sample. Since the chemical environment of thediffusable label should be known before wetting with sample, thediffusion rate of the diffusable label after wetting is influencedmainly by the constituents of the sample and the temperature, especiallywhen it is assumed that operations are carried out at approximatelynormal pressure.

Advantageously, diffusion of the diffusable label before wetting withsample is negligible, since the label is situated in an at leastvirtually dry environment in which there are no, or merely negligible,diffusion processes. For example, at least one first detector signal fordetermining a diffusable label concentration before wetting and at leastone second detector signal for determining a diffusable labelconcentration after wetting across a particular period and/or at aparticular time, can be used as a basis for detecting a diffusion of thediffusable label, after wetting of the label with sample in the form ofa diffusion rate of the diffusable label. Alternatively or additionally,it is also possible to observe a period only after wetting of thediffusable label. Further alternatively or additionally, a detectionsignal can be effected at a particular time after wetting of the labelwith sample. In some instances, the diffusion of the diffusable labeldepends on only few, previously known parameters. For example, thediffusion rate of the diffusable label used in a glucose determinationshould not depend on the concentration of the analyte to be determined,in this case glucose.

The piece of correction information thus includes at least one piece ofinformation about at least one interfering variable of the sample. Forexample, the interfering variable can, as explained above, be a Hctand/or a temperature of the sample or a combination thereof. Otherinterfering variables also are correctable.

If one or more detector signals are used for detecting diffusion of thediffusable label, the detector signal(s) can be compared with one ormore reference curves that are characteristic of various temperature andconcentration relationships of the sample. From this, it is possible todirectly or indirectly determine the temperature and/or at least oneother parameter of the sample. Alternatively or additionally, it ispossible, at different times after wetting of the diffusable label withthe sample, to detect concentrations of the diffusable label and tocompare them with stored reference curves. Alternatively oradditionally, it is possible, within particular time ranges after thewetting of the label with sample, to ascertain rises in the detectorsignal, which can likewise be compared with reference curves. In thismanner, it is possible to determine both the temperature-dependentinfluence of the diffusion rate of the diffusable label, and theinfluence of the diffusion rate of the diffusable label owing to thechemical composition of the sample.

As noted above, detecting the diffusable label can be carried out withthe same detector for detecting the analyte. The at least one detectorshould be able to carry out an optical detection at multiplewavelengths. Alternatively, it is possible to use a combined detectorthat has two detector units that can detect at different wavelengths. Ifdifferent detectors are used for the analyte detection and diffusablelabel detection (e.g., at least one analyte detector and at least onelabel detector, for example at least one optical analyte detector and atleast one optical label detector, the detectors can be arranged indifferent ways. For example, the detectors can be arranged on sides ofthe test element that oppose one other (e.g. the analyte detector on thedetection side and the label detector on the sample input side).Alternatively or additionally, the detectors can be completely or partlyarranged on the same side of the test element (e.g., on the detectionside). In addition, filters can be used in the beam path between lightsource and detector. Filters generally are able to filter out one ormore wavelengths of the light by absorbing or reflecting them.

The methods also include a step of detecting an analyte of interest in abody fluid sample with a test element as disclosed herein. As usedherein, “detection of a reaction of the test chemistry with the analyte”means a process in which the reaction itself and/or one or morereactants involved in the reaction and/or one or more reaction productsare acquired qualitatively or quantitatively by qualitative and/orquantitative acquisition of at least one change in property of thesample and/or of the test chemistry and/or of at least one furtherelement or range which is influenced by the detection reaction. Forexample, the reaction of the test chemistry with the analyte can bedetected by means of electrochemical and/or optical detection methods.Such detection methods, which can be carried out using at least onedetector are known to one of skill in the art.

In some instance, the body fluid sample is blood or a constituentthereof.

Thus, it is possible to ascertain at least one optical measurementvalue, such as at least one reflectance value, which detects thereaction of the test chemistry with the analyte. As noted above, it ispossible to ascertain at least one optical measurement value (e.g., atleast one further reflectance value), which detects the diffusion of thediffusable label. As such, optical measurement values for detecting thereaction of the test chemistry with the analyte and for detecting thediffusion of label can be acquired in particular at differentwavelengths.

Optically detecting the analyte can be achieved by using a detectorhaving at least one optical sensor configured to receive light and togenerate at least one corresponding signal. In addition, at least onelight source can be used to illuminate the test element and/or a partthereof and/or the sample and/or a part thereof and/or an optionalcorrection element and/or a part thereof, the optical sensor being usedto detect reflected and/or scattered and/or emitted light. Whenoptically detecting the analyte and/or diffusable label, it isadvantageous when the wavelengths of the incident light and/or of thedetected light are distinctly different so that a distinction betweenthe two signals can be made. As used herein, “distinctly different”means a deviation of the wavelengths to be detected for analytedetermination with respect to the wavelength to be detected fordetermining the label of at least about 20 nm, of at least about 30 nm,or of at least about 50 nm.

In some instances, the analyte is determined at a different wavelengthwith respect to the diffusable label. In this manner, the diffusablelabel detector can have at least one diffusable label detector lightsource, and the analyte detector can have at least one analyte detectorlight source, it being possible to configure the label detector lightsource to illuminate the test element with light of a differentwavelength with respect to the analyte detector light source. Forexample, the light of the diffusable label detector light source canhave a shorter wavelength than the light of the analyte detector lightsource or vice versa. Specifically, the analyte can be detected with theaid of light within a wavelength range from about 400 nm to about 1000nm, from about 500 nm to about 800 nm, from about 640 nm to about 680nm, or about 660 nm. In this connection, the label can be detected withlight within a wavelength range from <about 400 nm, from about 300 nm to<about 400 nm, or at about 360 nm.

An inverse design is also possible. That is, the diffusable label can bedetected with the aid of light within a wavelength range from about 400nm to about 1000 nm, from about 500 nm to about 800 nm, from about 640nm to about 680 nm, or at about 660 nm. In this connection, the analytecan be detected with light within a wavelength range from <about 400 nm,from about 300 nm to <400 nm, or at about 360 nm.

In some instances, the diffusable label absorbs light within awavelength range from about 400 nm to about 800 nm. The diffusablelabel, which can be in the form of a dye, can be excited with the aid oflight having a wavelength within a wavelength range from about 300 nm toabout 800 nm (but preferably less than 400 nm) or absorb light withinthe wavelength range, or from about 300 nm to about 500 nm, or fromabout 340 nm to about 380 nm.

In other instances, an inverse design can be used in which a differentlight source is used in each case for analyte determination anddiffusable label determination. For example, two different LEDs can beused. The LED for exciting the analyte can have an intensity maximumaround about 660 nm, whereas the LED for exciting the diffusable labelcan have an intensity maximum around about 360 nm. Alternatively, it isalso possible to use a light source having light that is processed withthe aid of optical means, such as filters, apertures or mirrors, so thatthe detection region is irradiated with light of different wavelengthsat different times. In some instances, the wavelength range of the lightfor detecting the diffusable label is at least about 5 nm, at leastabout 10 nm, at least about 50 nm, or at least about 100 nm away fromthe wavelength range of the light for detecting the analyte. This can berealized in various ways. Alternatively, the wavelength range of thelight for detecting the diffusable label is within a range from about 5nm to about 100 nm away from the wavelength range of the light fordetecting the analyte.

To determine the interval between the wavelength ranges for detectingthe diffusable label and the analyte, one can consider the intervalbetween the absorption maximum of the diffusable label and of theanalyte. For example, it is possible to use two light sources havingdifferent wavelength ranges of the emitted light. In addition, it ispossible to use only one light source having a broad wavelength range ofthe emitted light, it being possible to use filters in the beam pathwith the result of using the filters being that the determination of thelabel is carried out at a different wavelength with respect to thedetermination of the analyte.

In some instances, the methods use not only at least one firstdiffusable label but also at least one further diffusable labeldifferent from the first diffusable label, where the diffusion rate ofthe first diffusable label and the diffusion rate of the at least onefurther diffusable label are influenced by at least one first propertyof the sample and by at least one further property of the sampledifferent from the first property, respectively. By using more than onediffusable label, it is possible to determine possible influences on thediffusable label by different properties of the sample. The firstdiffusable label can be erioglaucine, indigo carmine, hydroxynaphtholblue, 1,1-diethyl-4,4-carbocyanine iodide and amaranth and can be usedto ascertain an influence of a first property of the sample on thediffusion rate of the diffusable label. For example, the first propertyof the sample can be a temperature of the wetted test field and/or ofthe sample, or a Hct of the sample to generate at least one first pieceof correction information. Likewise, the at least one further diffusablelabel can be erioglaucine, indigo carmine, hydroxynaphthol blue,1,1-diethyl-4,4-carbocyanine iodide and amaranth and can be used todetermine at least one further property of the sample, such as, forexample, temperature or Hct of the sample to generate at least onesecond piece of correction information. In some instances, use is madeof a combination of a first diffusable label such as a sulfone dye fordetermining the influence of temperature on the diffusion rate and afurther diffusable label such as an azo dye for determining a furtherinfluence of Hct of the sample on the diffusion rate. In otherinstances, a single label such as a sulfone dye is sufficient fordetermining the influence of a first and at least one further propertysuch as temperature and Hct of the sample on the diffusion rate of thelabel.

By using more than one diffusable label, however, an increased accuracyof the test method can be made possible. For example, two differentdiffusable labels can be used, where one of the labels exhibits adependence of the diffusion rate on the basis of temperature but only aslight dependence of the diffusion rate on the basis of Hct, and where asecond diffusable exhibits a dependence of the diffusion rate on thebasis of Hct but only a slight dependence of the diffusion rate on thebasis of temperature. It is thus possible to gain more accurate piecesof correction information for determining the concentration of theanalyte. It also is possible to use different diffusable labels havingdifferent spectral properties (e.g., dyes that absorb differently).

In some instances, the reaction of the test chemistry with the analyteis detected by means of at least one first detector, which can bereferred to as an analyte detector. Specifically, the analyte detectorcan include at least one first photosensitive detector element such asat least one first photodiode. In addition, the analyte detector caninclude at least one analyte detector light source such as at least onefirst LED.

The diffusion of the diffusable label can be detected by means of atleast one second detector, which can be referred to as a diffusiblelabel detector or diffusion detector. Specifically, the label detectorcan include at least one second photosensitive detector element such asat least one second photodiode. In addition, the label detector caninclude at least one label detector light source such as at least onesecond LED. As noted above, the first detector and the second detectorcan be formed separately or can also be at least partly identical. Moreparticularly, the analyte detector light source and the label detectorlight source can be formed differently, whereas the first photosensitivedetector element and the second photosensitive detector element can bedesigned to be identical in terms of components. For example, thedetection side of the test element can be provided with the analytedetector light source and the label detector light source forilluminating the test element or parts thereof, and also with a commonphotosensitive detector element such as a common photodiode, whichalternately receives reflected light of the label detector light sourceand of the analyte detector light source. Other designs are alsoconceivable in which different photosensitive detector elements areused.

As noted above, the analyte and/or the diffusable label can in each casebe independently detected electrochemically and/or optically. If, forexample, the analyte is to be analyzed electrochemically and thediffusable label optically, or the analyte optically and the diffusablelabel electrochemically, both at least one electrochemical and at leastone optical detector is generally necessary. Alternatively, both theanalyte and the diffusable label can be detected optically and/orelectrochemically. To this end, two different detectors can be used, andthis is especially advantageous when the wavelength ranges forascertaining the analyte concentration are very different from thewavelength ranges for ascertaining the label concentration. However, italso is possible to use one detector that provides the possibility ofdetecting light at more than one wavelength. If the analyte anddiffusable label are not situated within a common test chemistry layer,it may be advantageous to perform the detection of the analyte and ofthe diffusable label from different sides of the test element. To thisend, detecting the analyte would advantageously be carried out on theside facing away from the sample input side, whereas detecting thediffusable label can take place outside the test element such as, forexample, in a region of the test field closer to the sample input side.Alternatively, it is possible to use one or two detectors in the eventthat the diffusable label is not situated on or in the same test fieldas the detection reagents. For example, one detector can cover the areaof two test fields or two test elements. In this manner, the regionscontaining diffusable label can be illuminated with light of a differentwavelength with respect to the regions containing no label.

The concentration of the diffusable label and/or of the detectionreagent can be detected either directly or indirectly. In the case of adirect detection, use is made of a method that immediately qualitativelyand/or quantitatively detects the presence of the substance to bedetected, in this case the label or the detection reagent for theanalyte. In contrast, and in the case of an indirect detection, thepresence of the at least one diffusable label or of the one detectionreagent is qualitatively and/or quantitatively inferred via at least oneconceptual, theoretical or experimental intermediate step. For example,this can be done via the presence and/or formation and/or decrease ofone or more further substances, it being possible in turn toqualitatively and/or quantitatively infer the label or analyte, and sothe label or analyte can be detected indirectly. A known example of sucha detection reagent for the detection of blood glucose is nicotinamideadenine dinucleotide (NADH) (i.e., the reduced form of nicotinamideadenine dinucleotide (NAD⁺)), which can be directly detectedphotometrically. Altogether, for the design of possible labels ordetection reagents and test fields, reference can, however, be largelymade to the art.

As used herein, “conversion” means a reaction in which the analyte isinvolved and/or is chemically modified. In this manner, the testchemistry can ensure the specificity of the test element. For example,the detection can be achieved via a test chemistry containing an enzymeand a coenzyme by, for example, redox equivalents transferred by theanalyte through the coenzyme by a mediator, such as manganese dioxide.The redox equivalent can be converted for photometric and/or opticalconcentration measurements of the analyte with an indicator.

In some instances, the reaction of the test chemistry with the analyteis detected by means of at least one first detector, where diffusion ofthe diffusable label is detected by means of at least one furtherdetector such as at least one second detector.

As explained above, the methods generate at least one piece ofcorrection information from the diffusion of the diffusable label. Themethods therefore also include a step of correcting the analyteconcentration measurement with the at least one piece of correctioninformation that was obtained by detecting diffusion of the diffusablelabel in the actual sample and from the general relationship between theinterfering variable and the diffusion of the label. This can beachieved in a simple manner by using the detected diffusion of the labelin the actual sample (e.g., the optical measurement value of the labeland, more particularly, the reflectance of the label field), as afunction value of the general relationship as a function value of thecalibration curve or calibration function, producing the piece ofcorrection information.

The thus ascertained piece of correction information can then be usedfor correcting the analyte concentration and/or for correction of thedetection of the reaction of the test chemistry with the analyte. Forexample, in the sample in which the analyte concentration is to bedetermined, at least one uncorrected optical measurement value, forexample a reflectance, can be initially acquired, which is subsequentlycorrected with the piece of correction information in order to obtain acorrected optical measurement value. The correction can, for example, beachieved by addition or subtraction of the piece of correctioninformation from the uncorrected optical measurement value and/or bymultiplication of the piece of correction information with theuncorrected optical measurement value and/or by formation of a linearcombination from the piece of correction information and the uncorrectedoptical measurement value.

For example, the piece of information can be a piece of informationabout how at least one measurement value such as, for example, anelectrochemical and/or optical measurement value, is to be convertedinto an analyte concentration. For example, the piece of correctioninformation can include a piece of information about which conversionrule is to be applied when converting the at least one measurement valueinto the analyte concentration and/or how a conversion rule is to bedesigned and/or modified. As such, multiple pieces of correctioninformation corresponding to detected diffusion of the label can bestored in a data processing device and/or in a data storage device.

Alternatively or additionally, it is possible to store at least oneassignment rule which can be generated analytically, empirically orsemi-empirically, which assigns to each acquired diffusion a piece ofcorrection information for determining the analyte concentration. Forexample, the piece of correction information can be generated bycomparing a detector signal of a diffusable label concentrationdetermination with stored reference curves, by it being possible toassign a profile of the detector signal to a particular reference curve,which is in turn characteristic of a particular temperature and/or of aparticular Hct.

It is possible for further pieces of correction information to bedetermined through determining the diffusable label concentration. Forexample, it is possible to ascertain a moistness of the test elementbefore wetting with sample by comparing a detector signal for the labelconcentration before wetting with a reference value.

EXPERIMENTAL

The inventive concept will be more fully understood upon considerationof the following non-limiting examples, which are offered for purposesof illustration, not limitation.

Example 1 Methods and Materials

FIG. 1 a shows an exemplary test element 50 for detecting at least oneanalyte 117 in a sample 110, on which a test field 100 is arranged on atest support 60. The test element 50 is configured to be used in adevice 190 as shown in FIG. 1 d. The test field 100 includes a testchemistry 102 of one or more detection reagents, such as an enzyme thatconverts the analyte 117 and thus makes it possible to infer an analyteconcentration. In addition to the test chemistry 102, at least onediffusable label 104, 104′ also can be situated in or on the test field100; however, the diffusable label 104, 104′ can be situated on aseparate test element 50 or on another site on the test support 60 ofthe test element 50. The diffusable label 104, 104′ can be arrangedtogether with the test chemistry 102 and, in this way, form at least onetest chemistry layer 118. The test chemistry layer 118 also is known asa reactive layer owing to its reactivity. Alternatively, the diffusablelabel 104, 104′ can be arranged in a separate layer and form a separatelabel layer 120, as shown in FIGS. 1 b and 1 c.

Situated adjacent thereto is a pigment layer 122. The pigment layer 122can be at a distance from the test chemistry layer 118. Introduced inthe pigment layer 122 is, for example, a pigment such as TiO₂ or ZrO₂.In an optical detection, the pigment layer 122 serves, as shown in FIG.1 d, to reflect the incident light 150 b that penetrates through boththe test support 60 and the reactive layer 118. This ensures that thelight 150 b of the light source 140, while being able to interact withthe detection reagents in the reactive or test chemistry layer 118, isnot influenced by other constituents of the blood 110. These interferingconstituents, such as red blood cells, can be kept away from thereactive layer 118 by a partition layer 106.

FIGS. 1 b and 1 c each show a test field 100 on a test support 60 of atest element 50, as can be measured in the device 190 of FIG. 1 d. Thetest field 100 in FIG. 1 b shows the state of the test field 100 beforewetting 108 with a sample 110. In contrast, the test field 100 in FIG. 1c shows the state after wetting 108 of the test field 100 with sample110. In this connection, the sample 110 may be blood.

The diffusable label 104, 104′ can be situated in a first region 114 ofthe test field 100, in which the test chemistry 102 may likewise besituated. Alternatively, the test chemistry 102 can be accommodated in aseparate layer 118 or in a second region 116 of the test field 100. Ifthe diffusable label 104, 104′ is accommodated in a separate layer 120,this is known as a label layer 120, from which the test chemistry layeror reactive layer 118 is distinguished separately. If diffusable label104, 104′ and test chemistry 102 are accommodated in the same layer,this is known only as a test chemistry layer 118, 120.

In FIGS. 1 a-c, one or more further layers (not shown) can be presentabove, between or below the two layers 118 and 120. Furthermore, furtheradditives such as pigments, fillers, auxiliaries and other substancescan be present in both the test chemistry layer 118 and the label layer120, giving rise to a pigment layer 122. Alternatively, the additionalsubstances can be present in only one or in more than one of layers 118,120.

As shown in FIGS. 1 b-c, the diffusable label 104, 104′ spreads afterwetting 108 with sample 110 from the first region 114 of the test field100, in this case the test chemistry layer 118, into at least one secondregion 116. The second region 116 can be situated directly adjacent tothe first region 114 or be at a distance from the first region 114. Insome instances, the diffusable label 104, 104′ can diffuse through alllayers of the test field 100. The second region 116 also can be thesupernatant 112 of the blood 110 that has not been taken up by the testfield 100 and that has accumulated on the sample input side 107.Alternatively or additionally, the second region 116, into which thediffusable label 104, 104′ diffuses after contact with the sample 110and can be detected, can be outside the test element 50. Further layerscan be situated between the first 114 and second region 116, such as thepartition layer 106. Alternatively or additionally, the second region116 can be a component of the test element 50 and/or test field 100.

For detecting the diffusable label 104, 104′, one or both of regions 114and/or 116 can be used. If the first region 114 is used, “bleeding” ofdiffusable label 104, 104′ from the first region 114 is detected byoptical or electrochemical means. As used herein, “bleeding” means adiffusion of the diffusible label 104, 104′ from the first region 114.Depending on the diffusable label 104, 104′ used, this can lead to anincrease or decrease in the measurement signal. Alternatively, theinward diffusion of diffusable label 104, 104′ can be detected in partof or the entire second region 116.

The measurement signal increases or decreases during the diffusionprocess depending on whether the diffusable label is an absorption dyewhose reflection 160 a of incident light 150 a is detected or is afluorescent dye whose conversion of the incident light to anotherwavelength is detected. Conversely, the measurement signal will decreaseor increase when detecting the absorption dye or fluorescent dye takesplace in the second region 116. Alternatively, both regions 114 and 116can be used for detecting diffusion of diffusable label 104, 104′.

As an alternative to optically detecting diffusion of diffusable label104, 104′, the diffusable label can be electrochemically detected. Tothis end, an electrode accommodated in the first region 114 would detectthe bleeding of the first region 114 of diffusable label 104, 104′ afterwetting 108.

FIG. 1 d shows a device 190 having two light sources 130 and 140 withina housing 188, each on one side of a test element 50 inserted into thehousing. In the device 190, light 150 a can be irradiated from a firstlight source 130 onto the upper side of a test element 50 to be measuredin the device 190. The light 150 a emanating from the first light source130 strikes the surface of the test field 100 of the test element 50 andis reflected there. The light beam 160 a reflected on the test field 100is collected by a first detector 170. On the route from the first lightsource 130 up to the first detector 170, the light 150 a moves to someextent through the sample 110 and/or the supernatant of the sample 112,which supernatant can simultaneously represent a second region 116 ofthe device 190. In the process, light 150 a, 160 a can strike diffusablelabel 104, 104′, which has diffused from the first region 114 into thesecond region 116 after wetting 108 with sample 110 and now absorbs someof the incident light 150 a and/or reflected light 160 a.

Alternatively or additionally, the bleeding of diffusable label 104,104′ after wetting 108 of the test field 100 can be detected with asecond detector 180. This can be performed when diffusable label 104,104′ is introduced together with the test chemistry 102 in the testchemistry or reactivity layer 118. To this end, on the underside of thetest element 50, opposite the sample input side 107, light 150 b from asecond light source 140 is irradiated through the test support 60 andthe test chemistry 102 in the test chemistry or reactive layer 118 andreflected on the pigment layer 122. Light 160 b from the second lightsource 140 is reflected on the pigment layer 122 and is collected by thesecond detector 180. In this way, it is possible to detect both thelight for the detection reaction, which makes it possible to infer theconverted quantity of the analyte 117, and the light influenced bydiffusable label 104, 104′ or the influenced radiation, which reflectsthe diffusion of diffusable label 104, 104′.

If light source 140 and detector 180 are designed to emit or detect bothlight at the corresponding wavelength for detecting the detectionreagent of the test chemistry 102 and light at the correspondingwavelength for detecting diffusable label 104, 104′, one light source140 and one detector 180 can be sufficient for observing both thedetection reaction of the analyte 117, and the diffusion of diffusablelabel after wetting 108 of the test field 100.

Additionally, evaluation units 175 and/or 185 can be situated in oradjacent to detectors 170 and/or 180, which compare the signal withreference curves or reference values stored therein.

The detectors 170 and 180 used can be photodiodes, charge-coupled deviceCCD) sensors or complementary metal-oxide semiconductor (CMOS) sensors.Light sources 130 and/or 140 can be LEDs, mercury lamps or other lightsources having the desired wavelengths. As noted above, evaluation unit175 and/or 185 can be situated adjacently in or on detectors 170 and/or180. Evaluation unit 175 and/or 185 can be a microprocessor or anothersuitable calculator unit. However, it is possible for merely a storagedevice to be situated in the detector, which storage device can transferdata to an external evaluation unit (not shown here) or be read thereby.

Following the measurement, the test element 50 can be taken out againfrom the housing 188 so that a further test element 50 can be insertedfor a further measurement. The housing 188 serves especially asprotection for the electronic components within the housing 188, such asdetectors 170 and/or 180 with evaluation unit 175 and/or 185 and alsolight sources 130 and/or 140. However, housing 188 also serves asprotection for the user to prevent maloperation. The housing 188 has anopening 189 so that the test element 50 having the test field 100 can beinserted into the housing 188.

As an alternative to the arrangement of diffusable label 104, 104′within a restricted region of the test field 100, as shown in FIG. 1 b,it is possible for diffusable label 104, 104′ to be situated within theentire test field 100 of the test element 50, making it possible todetect bleeding of the test field 100 of diffusable label 104, 104′ withthe supernatant 112 serving as second region 116 following wetting 108of the test field 100.

An alternative arrangement of test chemistry 102 and diffusable label104, 104′ is shown in FIGS. 1 e-f. A test element 50 is shown that has atest support 60 on which a test field 100 is arranged and in which alabel layer 120 and a reactive layer 118 are arranged next to oneanother. Situated in the label layer 120 is diffusable label 104, 104′,which can be optionally separated from the reactive layer 118 by abarrier 101. In some instances, however, the test element 50 does nothave a barrier 101 between label layer 120 and reactive layer 118. Theresult of using the optional barrier 101 can be that before and/or afterwetting of the test field 100 with sample 110, hardly any substanceexchange takes place between label layer 120 and reactive layer 118.After wetting of the test field 100, it is usually acceptable that asubstance exchange by diffusion can take place. The result of using thisarrangement of the label layer 120 next to the reactive layer 118 isthat these two layers can be independently illuminated by, in each case,light source 130 and 140. For example, the label layer 120 isilluminated by incident light 150 a of the first light source 130, andthe reactive layer 118 is illuminated by incident light 150 b of thesecond light source 140. The label layer 120 and the reactive layer 118can have an identical or non-identical volume and/or an identical ornon-identical layer thickness. FIG. 1 e shows a design in which thereactive layer 118 and the label layer 120 differ in layer thickness,whereas FIG. 1 f shows a design in which the reactive layer 118 and thelabel layer 120 are substantially identical in layer thickness. In someinstances, the label layer 120 and the reactive layer 118 aresubstantially identical in layer thickness and/or are substantiallyidentical in volume. As used herein, “substantially identical” meansidenticalness or else a deviation of the layer thicknesses or of thevolumes from one another by not more than about 20%, by not more thanabout 10%, or by not more than about 5%.

After wetting of the test field 100 with the sample 110, diffusablelabel 104, 104′ can diffuse from the first region 114, such as at leastpart of the label layer 120, into the second region 116. The secondregion 116 can include the supernatant 112 of sample 110 and/or afurther layer 116 above and/or lateral to (not shown here) the labellayer 120. If the barrier 101 is designed so that it is permeable todiffusable label 104, 104′, the reactive layer 118 can serve as at leastpart of the second region 116 into which diffusable label 104, 104 candiffuse after wetting. Conversely, part of the analyte to be detectedcan be situated in both the supernatant 112 and the second region 116.

In some instances, the region into which diffusable label 104, 104′ candiffuse and the region into which the analyte 117 can diffuse are aboutthe same size. In FIG. 1 e, the proportions of the layers 116 and 118are not necessarily portrayed true-to-scale for all conceivableembodiments having label layer 120 and reactive layer 118 arranged nextto one another. For instance, the second region 116 together with thefirst region 114 or the label layer 120 can have the same layerthickness and/or the same volume as the reactive layer 118. However,alternative designs also are contemplated. For example, in onealternative design, the label layer 120 can by itself have the samelayer thickness and/or the same volume as the reactive layer 118 (see,FIG. 1 f). Likewise, and for detecting reflected light in the form ofthe reflected light of the first light source 160 a and of the secondlight source 160 b, detector 170 can be used. Additionally, it ispossible to use a further detector, thus making it possible to collectthe reflected light beams 150 b and 160 b separately.

FIGS. 2-9 show curve profiles or kinetics for the outward flow ofvarious diffusable labels (e.g., dyes) from a label layer and testchemistry layer 118, 120 in a conventional test field 100 into thesupernatant of sample 112. In this connection, diffusable label 104,104′ is situated in the test chemistry layer 118 before wetting 108 ofthe test field 100. The compositions of the layers 118 are specified inTables 1 and 2 below. The curves in FIGS. 2-9 were detected inreflectance. In this connection, detection in reflectance or detectionof a reflectance value generally refers to an optical measurement basedon reflection of waves, especially light or reflection of diffuse,undirected waves. Reflectance can be detected in various ways such as,for example, at least one signal of a detector that detects reflectedlight being acquired. As indicated in FIGS. 2-9 on the vertical axis,reflectance can be simply specified as signal I in arbitrary units(symbolized by [-]). Alternatively, it is possible for the reflectanceto be specified in other units, for example in the form of asurface-based measure of the reflectance (i.e., a degree ofreflectance). It also is possible to specify a ratio of reflected energyto incident energy in percent, a so-called albedo value.

In the experiments, the layers were excited at 660 nm with a measurementinstrument (e.g., a Gen5Red measurement instrument) having a LED thatemits light at a wavelength of 660 nm and detected in reflectance with aBPW34 silicon detector. For measuring behavior of the various dyes, usewas made of test elements 50 having test fields 100 on a test support 60as shown in FIGS. 1 a-d. In some experiments, a further test chemistrylayer 118, 120, which contained not only the detection reagents but alsoa varying percentage of dye (i.e., 0.05% or 0.1%) was applied to thereactive layer 118. The former is hereinafter called the label layer120.

Two different layers, the composition of which is specified in Tables 2and 3, were applied to a Pokalon film serving as test support 60 bydoctor blading at a rate of about 5 m/min. The Pokalon film of theN332EM type from Lonza (Germany) is matt on one side, corona-treated andabout 140 μm thick. In the recipes of the test chemistry layer 118 andthe label layer 120, use was made of glucose dehydrogenase together withthe coenzyme carba-NAD (cNAD). This was measured photometrically at 360nm from the side opposite the sample input side with the BPW34 silicondetector. The diffusion of diffusable label 104, 104′ was measured onthe underside of the test field, (i.e., opposite the sample input side)with the aid of a light-emitting diode and a detector.

Dyes were selected, the absorption maximum of which is distinctlydifferent with respect to the wavelength at which the glucose reactionis detected. In addition, the detection reagents were selected so as notexhibit any appreciable absorption. Furthermore criteria for theselected diffusable label were water-solubility and good chemical andlight stability and availability.

Table 1 below specifies the diffusable labels (e.g., dyes) investigated,with their structural formulae and absorption maxima in varioussolvents.

TABLE 1 Name, structural formula and absorption maximum (in the mediaindicated within parentheses). Dye Structure Absorption Erioglaucine

625 nm (H₂O) Indigo carmine

608 nm (H₂O) Hydroxynaphthol Error! Objects cannot be created 650 nm(MeOH/H₂O) blue from editing field codes. 1,1-Diethyl-4,4- carbocyanineiodide (1260)

703 (648) nm (MeOH) Amaranth

521 nm (H₂O)

These dyes were added in different ratios as dry substance to the testfields 100 of the test elements 50 of FIGS. 2-9. The proportion of dyeis indicated in the individual figure descriptions.

The makeup of the test chemistry layer 118 and label layer 120 for thesubsequent experiments is specified in both Tables 2 and 3 below.

TABLE 2 1^(st) test chemistry layer. Subst. W-Rec Rec Actual Sd spDa S(manufacturer) Conc g/m² g/100 g g MR g g/m² 1 Thickener 7% 19.961 14.84103.93 H₂O 15.606 11.61 806.9 0.00 Gantrez S97 1.397 1.04 70.0 1.3970.37 (ISP Corp.; New Jersey) NaOH, 16% pH = 6.8 2.958 2.20 123.1 0.4730.13 Suspension 33.598 24.99 175.10 5 Water, dd. 30.60.152 22.76 182.200.00 6 Sipernat FK 3.00 2.23 17.80 2.998 0.80 320DS (Evonik) Wettingagent/ 12.88 9.58 67.12 other 2 Mega 8 0.300 0.22 1.56 0.300 0.08 3Geropon 0.040 0.03 0.210 0.040 0.01 7 Propiofan- 8.044 5.98 41.90 4.0221.07 70VAR (BASF) 4 PVP 25000 0.999 0.74 5.20 0.999 0.27 (Fluka) 8 K—PO₄buffer,   1M 3.50 2.60 18.25 0.483 0.13 pH 6.8 (Merck) 9 Enzyme 68.0350.59 354.50 solution K—PO₄ buffer, 0.1M 31.0 23.05 177.50 0.43 0.12 pH6.8 (Merck) cNAD-Na 5.98 4.45 34.2 5.98 1.60 (Roche) GlucDHmut2 #4 3.0/2.23/ 17.2 3.00 0.80/ (kU/g)/(kU/m²) 915.00 680.46 244.14 (Roche) Water,dd. 28.0 20.86 160.6 0.24 pH before 6.8 adjustment pH after 6.77adjustment NaOH, 16% for 0.000 fine adjustment Total 134.47 100.00700.65 41.09 18.96 S: sequence; Subst: substance; MR: master ratio;Conc: concentration; W-Rec: working recipe; Rec: recipe; Sd: solid;spDa: specific dry application

The substances were applied to the Pokalon film of a test field of atest support in the sequence specified. Subsequently, the layers weredried at about 50° C. for about 15 min.

TABLE 3 2^(nd) test chemistry layer. W-Rec Rec Actual Sd spDa S Subst.Conc g/m² g/100 g g PS g g/m² Sy 69.503 71.66 537.37 2 K—PO₄, pH = 6.81M 8.800 9.07 68.04 1.25 0.58 3 ZrO₂ (FCM) 35.551 36.65 274.88 35.5516.40 1 Water, dd. 25.152 25.93 194.45 Thickener 7% 23.329 24.05 180.354 H₂O 18.824 19.41 806.9 0.00 0.00 Gantrez S97 1.633 1.68 70.0 1.63 0.75NaOH, 16% pH = 6.8 2.872 2.96 123.1 0.46 0.21 Wetting agent/other 4.1654.29 32.17 5 Mega 8 0.235 0.24 1.82 0.24 0.11 6 Propiofan 3.930 4.0530.35 1.97 0.91 7 NaOH, 16% 0.00 0.00 pH before adjustment: 6.7 Total96.997 100.00 749.89 41.09 18.96 S: sequence; Subst: substance; Sy:slurry; Conc: concentration; W-Rec: working recipe; Rec: recipe; PS:partial solutions; Sd: solid; spDA: specific dry application

The substances were applied to the 1^(st) layer on the test field of theabove test support in the sequence specified. The application took placeby means of a doctor-knife blade, which evenly applies theaforementioned chemicals to the test support by means of doctor bladingover table. The particular dye was applied to the test support as thelast step. The coating took place at room temperature and a relative airhumidity of about 43%. Subsequently, the layer was dried at about 50° C.for about 20 min.

The buffer solutions used were prepared according to Table 4.

TABLE 4 Buffer solutions. g Gantrez solution, 7%, pH = 6.9 Water, dd.806.9 Gantrez S97 533448 70.0 7.0% NaOH, 16% 123.1 1000.00 6.92 pH (6.8+/− 0.1): Silicic acid suspension (water, dd.) Silicic acid, SipernatFK320DS 533235 17.8 8.9% Water, dd. 182.2 200 K₂HPO₄ solution, 1M (fillup to 500 174.18 g/mol mL up to calibration mark) K₂HPO₄(03593622001/#51833200) 87.10 15.4% Water, dd. 478.60 565.70 KH₂PO₄solution, 1M (fill up to 500 136.09 g/mol mL up to calibration mark)KH₂PO₄ 68.05 12.5% Water, dd. 476.76 544.81 K phosphate buffer, pH =6.8, 1M 13.8% Initially charged K₂HPO₄ solution 400.00 (15.4%) KH₂PO₄solution (12.5%) 494.80 Total buffer at pH 7.0 894.80 pH (6.8 ± 0.1):6.8 K phosphate buffer, 0.1M, pH = 6.8 (fill up to 1 L up to calibrationmark) K phosphate buffer, 1M, pH = 6.8 100 Water, dd. 900 1.40% 1000 pH(6.8 ± 0.1): 6.8

Test elements having the dyes from Table 5 were prepared in a manner asexplained above and tested in experiments.

TABLE 5 Composition of the test fields in the various experiments (asdescribed and shown in FIGS. 2-9). 2nd layer 1st layer (66 μm), final pH6.75 (58 μm) V1 Standard Standard V2 Standard + 0.05% by weight ofamaranth Standard (17.9 mg/m²) V3 Standard + 0.1% by weight of amaranthStandard (35.8 mg/m²) V4 Standard + 0.05% by weight of erioglaucineStandard (17.9 mg/m²) V5 Standard + 0.1% by weight of erioglaucineStandard (35.8 mg/m²) V6 Standard + 0.05% by weight of hydroxynaphtholStandard blue (17.9 mg/m²) V7 Standard + 0.1% by weight ofhydroxynaphthol Standard blue (35.8 mg/m²) V8 Standard + 0.05% by weightof indigo carmine Standard (17.9 mg/m²) V9 Standard + 0.1% by weight ofindigo carmine Standard (35.8 mg/m²) V10 Standard + 0.05% by weight of1,1-diethyl-4,4- Standard carbocyanine iodide (1260) (17.9 mg/m²) V11Standard + 0.1% by weight of 1,1-diethyl-4,4- Standard carbocyanineiodide (1260) (35.8 mg/m²)

The dye was added as a dry substance to the 1^(st) layer beforeapplication to the test field and homogenized with a paddle stirrer(about 200 rpm), which was subsequently sieved and centrifuged.

All the dyes exhibited a temperature-dependent diffusion (e.g., an“outward flow”) into the upper blood, which was reflected in an increasein the reflectance signal as the concentration of the absorbing dye inthe label layer 120 was reduced. The respective magnitude of thedependence depended on the structure of the particular dye.

The example described herein concerns kinetics of erioglaucine, whichexhibited the strongest temperature dependence and were not dependentupon glucose concentration.

FIG. 2 shows various curve profiles of diffusion kinetics oferioglaucine within the test field before and after wetting of the testfield with sample (in this case, blood) on the basis of three differentfamilies of curves 204, 206 and 208 with the intensity of the reflectedlight being measured. Light of wavelength 625 nm was irradiated onto thetest field. The test field includes 0.05% erioglaucine as label in thefirst region 114 of the test field 100 shown in FIG. 1 b. Light ofwavelength 625 nm was guided by the underside of the test field 100directly onto the label layer 120, and the detector recorded reflectedlight from this test field side.

Various test fields were subjected to a temperature-dependentmeasurement, by blood having two glucose concentrations applied to thelayer and being measured at 660 nm. In the curve profile, it is possibleto distinguish three different families of curves, where the firstfamily of curves 204 exhibits a reflection of the test field at 5° C.and two different glucose concentrations (e.g., 0 mg/dl 204 a and 550mg/dl 204 b) in the sample. The second family of curves 206 was measuredat 25° C. either with 0 mg/dl glucose supplement 206 a or 550 mg/dlglucose supplement 206 b, whereas the third family of curves 208 wasmeasured at 45° C. One part of the curves was generated with a samplewithout addition of glucose 208 a, whereas a second part was ascertainedwith an addition of 550 mg/dl glucose 208 b.

As shown in FIG. 2, the slope of the kinetics is strongly dependent onthe temperature − at 5° C., the outward flow of the dye is distinctlyslower than in the case of 25° C., and the rate is in turn slower thanat 45° C. With the concentration of the dye in the label layer, it ispossible to influence the level and the speed of the end signal. Thequantity of analyte (i.e., glucose) had no influence on the diffusion ofthe label and vice versa, as can be seen from the curve profiles.

As shown in FIG. 3, similar results were achieved with a concentrationof 0.1% erioglaucine in the label layer 120 in the test field 100. Heretoo, it was possible to distinguish three families of curves 204, 206and 208, which were determined for the temperatures 5° C., 25° C. and45° C. for the first family of curves 204, the second family of curves206 and the third family of curves 208, respectively.

In both FIGS. 2 and 3, it is not possible to distinguish the differentglucose concentrations from one another at the respective temperatures,and this is shown by the subgroups 204 a, 206 a and 208 a for 0 mg/dlglucose and the subgroups 204 b, 206 b and 208 b having in each case 550mg/dl glucose. From these curve profiles, it can be deduced that,although the diffusion of erioglaucine is temperature-dependent, it isnot dependent on the analyte concentration. For this reason, this dye issuitable for inferring the temperature of the sample on the basis of itsdiffusion rate.

As shown in FIG. 4, it was possible to demonstrate the inventive conceptnot only for erioglaucine but also for hydroxynaphthol blue. Here too,the dependence of the diffusion rate of the label was measured atdifferent temperatures −5° C. for the family of curves 204, 25° C. forthe family of curves 206 and 45° C. for the third family of curves 208,at different glucose supplements. The basic finding was that, while thediffusion rate of hydroxynaphthol blue is temperature-dependent but notdependent on the analyte concentration.

As shown in FIG. 5, it was possible to additionally show that thediffusion rate of erioglaucine was dependent not only on the temperaturebut also on Hct of the sample. To this end, reflection curves wererecorded at different temperatures −5° C. for the family of curves 210,25° C. for the family of curves 212 and 45° C. for the family of curves214. In this connection, which profile the curve takes depends on Hct ofthe sample. For instance, curves can be distinguished at 5° C. in termsof their kinetics, depending on whether they have blood as sample, asshown for the family of curves 210 b, or whether, as for the family ofcurves 210 a, the sample consists of water. Likewise, the family ofcurves 212 can be distinguished in terms of its profile, on the basis ofcurves for blood samples 212 b at 25° C. and curves for water samples212 a at 25° C. The other family of curves 214 at 45° C. can bedistinguished according to the samples of water having the curve 214 aand blood having the curves 214 b. As can be seen, the label diffusesmore rapidly out of the test field 100 at elevated temperature. Inaddition, a varying diffusion rate at the respective temperatures can bedistinguished for varying Hct. For example, at the same temperature of5° C., a distinctly higher diffusion was found at lower Hct. On thebasis of these curve profiles, it is consequently possible to infertogether the influence of temperature and Hct. If the temperature isknown, it is possible, on the basis of the diffusion of diffusablelabel, to infer the Hct, and vice versa.

To further investigate the influence on the measurements by Hct, thetest chemistry layer 118 containing diffusable label 104, 104′ in theform of erioglaucine was investigated, particularly the outward flow oferioglaucine from the layer.

FIG. 6 shows curve profiles of three different samples having varyingHct, the test field 100 being coated with 0.05% erioglaucine. The curvesof the family of curves 204 have a Hct of 65%, whereas the curves of thefamily of curves 206 have a Hct of 45%, and the curves of the family ofcurves 208 a Hct of 25%. In this connection, the applied sample did notcontain any glucose. Here, the samples are blood samples brought to 0mg/dl glucose by rolling at room temperature, sample movement wasgenerated by the rolling and practically complete degradation of anyglucose present taking place overnight owing to the cells contained inthe sample.

FIG. 7 shows families of curves 204, 206 and 208 having respectively65%, 45% and 25% Hct in blood were recorded, samples having differentglucose concentrations being found in the families of curves. For eachfamily of curves 204, 206 and 208, glucose concentrations of 0, 90, 150,350 and 550 mg/dl were recorded. On the basis of these families ofcurves, it likewise can be seen that diffusion of the diffusable labelwas approximately independent of the concentration of analyte 117 in thesample, but that there was a dependence of the kinetics on Hct.Consequently, Hct of the sample can be determined from the curveprofile.

Alternatively, it is also possible at a particular time, as indicatedhere after 5 seconds, to ascertain the reflection difference in relationto time 0, corresponding to the time of sample input, and to ascertaintherefrom the Hct of the sample.

FIG. 8 shows the mean values for the respective glucose concentrationsof 0, 90, 150, 350 and 550 mg/dl from the families of curves 204, 206and 208 from FIG. 7. This clarifies the independence of the curveprofiles from the glucose concentration of the sample.

FIG. 9 shows three families of curves 204, 206 and 208, and within thefamily of curves 204, a curve 216 for a test field was doped with 0.05%erioglaucine, whereas the curve 222 originates from a test fieldcontaining 0.1% erioglaucine. Both curves were recorded with an aqueoussolution and a Hct of 65%. The family of curves 206 was recorded with aHct of 45%, and the family of curves 208 with a Hct of 25%. Also in thecase of the family of curves 206, a curve 224 having 0.05% erioglaucinecan be distinguished from a curve 226 having 0.1% erioglaucine, whereasin the family of curves 208, a curve 218 having 0.05% erioglaucine and acurve 220 having 0.1% erioglaucine can be distinguished. Generally, themeasurements were stopped after 4 to 5 seconds to realize as short ameasurement time as possible.

From the families of curves 204, 206 and 208, which each include twodifferent diffusable label concentrations, it can be seen in the case ofthe line drawn at 5 seconds after sample input that a distinctionbetween the two concentrations of 0.1 and 0.05% of erioglaucine canbarely be distinguished. This means that even in the event of a decreasein diffusable label in a test element over the service life, anyrestriction of the correction means need not be feared.

Altogether, it can be observed that all the dyes investigated in FIGS.2-9 exhibited a temperature-dependent outward flow into the supernatantfrom blood after wetting of the test field. This becomes apparent in anincrease in the reflectance signal, since the concentration of theabsorbing dye is lowered in the test layer. The particular magnitude orthe particular extent of the dependence on the temperature depends onthe structure of the particular dye.

FIG. 10 shows the behavior of an analyte signal of a sample, in thiscase glucose in blood, at different temperatures and different Hct.Here, the analyte signal is plotted in FIG. 10 on the vertical axis andidentified by c and specified in mg/dl. As used herein, “analyte signal”means any desired measurement value or any desired measurement variableor variable derived from a measurement that reflects the analyteconcentration or that correlates with the analyte concentration or thatmakes it possible to infer the analyte concentration. The analyte signalcan, as an alternative to the specification in mg/dl, also be specifiedin other units such as, for example, in units of a detector signal inreflectance values or similar units. In FIG. 10, the analyte signal wasacquired at a time of 4 seconds after wetting of the test field with thesample, and this is symbolized as “@ 4 s”. As noted above, the values ofthe analyte signal shown in FIG. 10 are not measurement values, butrather schematic, fictitious values reflecting a typical profile of theanalyte signal.

FIG. 10 also shows, diagrammatically, dependencies of the analyte signalon two different influences of varying properties of the sample − thetemperature T, specified in ° C., and a Hct value, specified in percent.These properties are shown on the horizontal axis. In this connection,fictitious analyte values which, for example, could be acquired as partof a series of measurements with variation of the temperature T shown asfilled-in circles and identified by the reference number 234. Analytevalues that could be acquired as part of a series of measurements withvariation of the Hct are shown as open circles and identified by thereference number 236.

The values shown in FIG. 10 could, for example, be acquired with ameasurement assembly in which the sample is applied from an applicationside to a test field containing the test chemistry and optionally thelabel. The test field is irradiated with an analyte detector lightsource by means of a first LED from a detection side of the test fieldthat is opposite the application side, and light scattered on the testfield acquired by means of a photosensitive detector such as, forexample, a photodiode, to obtain a reflectance value. The reflectancevalue of the analyte measurement can then be converted by means of aknown, fixed rule into the analyte concentration (uncorrected),specified in mg/dl or in other concentration units. The analyte detectorlight source can be adapted in terms of its spectral properties in sucha way that it has a wavelength that is absorbed by a dye of the testchemistry. For example, the analyte detector light source can emit at awavelength of 360 nm.

The profile of the points for variation of the temperature 234 shows alowering of the measurement values for the analyte glucose with sinkingtemperature. This means that the user of a conventional test elementwithout temperature correction receives a higher analyte measurementvalue when the temperature is higher than during the calibration and alower value when the temperature during the measurement is lower thanthat during the calibration of the system. It is likely that thisbehavior is caused by a lowering of the temperature generally inhibitingin particular diffusion behavior, and so the analyte to be detectedadvances more slowly toward the test chemistry than at a highertemperature. In addition, the actual detection reaction for detectingthe analyte may be slowed down.

An inverse behavior was exhibited by the measurement signal with regardto Hct in the sample. As shown in FIG. 10, on the basis of themeasurement points for Hct 236, that the measurement signal for glucosedecreases when the Hct increases. The measurement signal for glucosedecreased within the temperature range of 5° C. to 45° C. to the sameextent as in the case of a rise in Hct from 25% to 65%. It is likelythat the cause of this behavior is likewise to be found in an inhibitionof diffusion processes by the increased Hct.

Both aforementioned effects are generally not directly separable, butthis is irrelevant for a correction. For example, it is generallypossible to achieve a correction for a combination of a particular Hctat a particular temperature without Hct and temperature being known.Alternatively, it would, however, also be possible to independentlydetermine one of these interfering variables, such as temperature andHct (i.e., independently of the diffusion of label). For example, itwould be possible to use at least one temperature sensor that candetermine an ambient temperature and/or a temperature of the testelement and/or a temperature of the sample (generally in the context ofthe present disclosure, no distinction is generally made between thesetemperatures). With knowledge of the temperature, it is then possible toachieve a correction by means of the ascertained label diffusion for aparticular Hct.

As will be explained in more detail below, the correction of the analyteconcentration can be carried out so that an analyte concentration at,for example, 21° C. or 25° C. (room temperature) and a Hct of 40% isascertained as corrected values of the analyte concentration, and socorrected analyte concentrations acquired under different conditions(temperature, Hct) can be compared with one another.

By contrast, FIG. 11 shows the influence of the temperature and/or Hcton a diffusable label signal. Again, fictitious values were plotted,which show symbolically a typical profile of a label signal. It would bepossible to acquire the values of the label signal with a measurementassembly analogous to the measurement assembly described above inrelation to FIG. 10 or with the same measurement assembly. For example,it would be possible to apply the sample from an application side to afield containing the label and optionally also the test chemistry, alabel field and/or a test field. The test field can be irradiated with alabel detector light source by means of a second LED from a detectionside of the test field that is opposite the application side, and bymeans of a photosensitive detector such as, for example, a photodiode.Light scattered on the label field and/or test field can be acquired toobtain a reflectance value R. In this connection, the label detectorlight source can be adapted in terms of its spectral properties so thatit has a wavelength that is absorbed by a label. For example, the labeldetector light source can emit at a wavelength of about 660 nm.

FIG. 11 shows the reflectance behavior R (specified on the vertical axiswithout units) of erioglaucine, which exhibits the same behavior in thecase of temperature change as in the case of Hct change within aparticular range of this parameter. In this connection, the reflectanceR can be acquired on a label field as a function of time after wettingof the label field or test field with the sample. After 4 sec, themeasurement can be stopped, and the reflectance value R recorded at thistime.

FIG. 11 also shows that the reflectance of the label increases uponlowering of the temperature from 45° C. to 5° C., as strongly as theincreasing of Hct from 25% to 65%. Again, it is thus possible to recordtwo effects that can be attributed to the two interfering variables,temperature and Hct. In the case of increasing temperature anddecreasing Hct, the diffusion of the label into the sample is favored,and so the staining of the label field and/or of the test fieldcontaining the label, as viewed from the detection side, decreases,resulting in the reflectance increasing. Conversely, in the case ofdecreasing temperature and increasing Hct, the diffusion of the labelinto the sample is inhibited, and so the staining of the label fieldand/or of the test field containing the label is preserved and thereflectance thus remains low. For example, the reflectance can again beacquired at a time of 4 s after application of the sample or at anotherpredefined time after application of the sample. Again, both effects aretypically separable only with difficulty. However, since the reflectanceduring the measurement of label is dependent on both interferingvariables, the reflectance during the measurement of label, which is atleast approximately independent of the analyte concentration, can allowjointly a total correction for both interfering variables.

FIGS. 12 to 14 show an alternative method of correcting a glucosemeasurement by a reflectance measurement of a label field. For thesemeasurements, 10 to 50 samples having different glucose concentrationsbetween 30 mg/dl and 550 mg/dl were measured optically, with reflectancemeasurements being carried out and subsequently the reflectance valuesbeing converted into corresponding glucose concentrations in aconventional manner (i.e., in a manner known to one of skill in the artwithout taking into account the Hct and the temperature). In thisconnection, 10-15 measurements were carried out in each case on sampleshaving a predefined Hct, with measurements being carried out on sampleshaving three different Hct values—in each case 10-15 measurements onsamples having 25% (identified in FIGS. 12 to 14 by reference number228), 10-15 measurements on samples having 45% (identified in FIGS. 12to 14 by reference number 230), and 10-15 measurements on samples having65% (identified in FIGS. 12 to 14 by reference number 232).

Plotted on the vertical axis in FIGS. 12 to 14 is “system error,” whichis identified by “E”. In glucose measurement instruments, the systemerror is a variable known to one of skill in the art. The system erroris specified in absolute units in the case of glucose concentrations upto 100 mg/dl, and in percent in the case of glucose concentrations above100 mg/dl. The system error is calculated in each case from the measuredvalue and the actual value of the glucose concentration, which, asexplained above, is influenced by the diffusion of the glucose in thesample.

The actual value of the glucose concentration can be determined by meansof a known laboratory method and/or by a known starting weight ofglucose in a sample during preparation of the sample. To calculate thesystem error E, the deviation of the measured value from the actualvalue is determined by means of optical reflectance measurement on atest field. In the case of measured concentrations of up to 100 mg/dl,the absolute deviation is specified as system error. In the case ofmeasured concentrations above 100 mg/dl, the deviation with respect tothe actual concentration is expressed as a ratio.

FIG. 12 shows the reflectance R of a label field specified on thehorizontal axis, which reflectance is influenced by the diffusion of thediffusable label in the sample. The diffusable label was erioglaucine,and reflectance was acquired 4 s after application of the sample to thelabel field or test field, analogously to the measurements in FIG. 11.Reflectance is specified here in percent, based on an absolutereflectance. Alternatively, it would be possible to plot otherrepresentations of results of a measurement of label on this horizontalaxis.

FIG. 12 thus shows that the family of points exhibit a correlationbetween the system error E of the glucose measurement and thereflectance behavior of the label, which can be utilized for correction.The correlation can be described by a line, more particularly a standardcurve, having the linear equation E=m·R+b, where the parameters m(slope) and b (axis intercept) can be determined from an alignment toexperimental values, according to established methods. For example, thiscan be achieved by a so-called fit. By means of the parametersdetermined in this manner, it is subsequently possible to correctanalyte measurements by firstly determining uncorrected values of areflectance and/or an analyte concentration in the case of an analytemeasurement, which values are then corrected by means of a correctionfactor corresponding to the known parameters of the correlation. Thus, acorrection of the analyte measurement to a corrected reflectance and/ora corrected analyte concentration at 25° C. and 40% Hct can take place.

The correlation in FIG. 12 thus shows that the influence on the glucosemeasurement by Hct correlates with the influence on the labelreflectance by the Hct. Exemplarily, a linear correlation was adoptedfor the correction of this influence, though more complex correlationsalso are possible. To quantify this correlation, a fitted line 238 wasaligned to the measurement values in FIG. 12. The fitted parameters ofsaid fitted line 238 the can be utilized to correct, after acquiring thereflectance of the label field, corresponding measurement values of theglucose concentration. This is shown in FIGS. 13 and 14.

FIG. 13 shows system error E for various Hct values in blood without useof the abovementioned piece of correction information as a function ofHct. It can be seen that, owing to the uncorrected measurement points,the system error scatters from about −22% in the case of a Hct of 65%right up to almost 20% in the case of a Hct of 25%.

By contrast, if these measurement values are corrected with the aid ofthe piece of correction information from the fitted lines 238, thescattering of the system errors can be drastically reduced. As shown inFIG. 14, the same measurement values were taken as basis, which werealso taken as a basis of the measurement in FIG. 13. However, themeasurement values were firstly corrected with the piece of correctioninformation from the fitted lines 238 and subsequently the system errorE was ascertained.

The system error can, for example, be described with the aid of a linearequation:

E=m*R+b  (1),

where E is the system error, m the slope of the lines, R the reflectancevalue and b the y-axis intercept of the lines.

With the aid of the error ascertained in this manner, it is possible toconvert a later-ascertained, uncorrected glucose measurement value intoa corrected glucose measurement value. The following arises:

gc _(corr) =gc _(uncorr) −E=gc _(uncorr) −m*R−b  (2),

where gc_(corr)=corrected glucose measurement value;gc_(uncorr)=uncorrected glucose measurement value; E=system error,m=slope of the lines, R=reflectance value; b=y-axis intercept.

The corrected results in FIG. 14 show that an efficient adjustment ofthe influence of the interfering variables temperature and Hct can beachieved by means measuring diffusable label and the correlation knownowing to the measurement in FIG. 12. For instance, following thecorrection in FIG. 14, all families of points 228, 230 and 232, havingrespectively 25%, 45% and 65% Hct, are approximately within a band ofabout ±10% or ±10 mg/dl system error. This means a halving of thescattering of the system error with respect to the non-corrected valuesfrom FIG. 13, and this distinctly increases the reliability of themeasurement for the user.

Example 2 Obtaining a Piece of Correction Information for the Correctionof an Analyte Measurement and Example of a Correction

An example of a correction of a glucose measurement on the basis of apiece of correction information generated from a diffusion of label isdescribed exemplarily below. In this connection, it is generallyapplicable that the reflectance R is a function F of the temperature Tand Hct both in the case of measurement of the label and in the case ofmeasurement of an analyte signal, and in both cases the same dependenceemerges:

R[%]=F(T,Hct)  (3).

Since the influences of the individual interfering variables oftemperature and of Hct generally form a joint interfering variableT×Hct, since in many cases the influences of temperature and of Hct arenot to be separated but to be corrected together and since theinfluences of temperature and of Hct on the measured reflectance R aregenerally contrary (see, e.g., FIG. 11), the following can be applied:

R[%]=F(T×Hct)  (4).

The function F represents a calibration curve that can be ascertainedfrom a measurement of label analogously to the measurements of adiffusion of label that were described above in connection with FIGS.11-13. For example, a series of measurements can be recorded analogouslyto the measurements in FIG. 11, and the calibration curve can bedetermined therefrom. As can be seen from the graph in FIG. 11, thecalibration curve F can be described in many cases as a line, having theequation:

F(R _(Label))=a+m·R _(Label)  (5).

In this connection, a represents an axis intercept or offset, whereas mdescribes a slope of the calibration curve. R_(Label) represents ameasured reflectance of the diffusable label at a particularconcentration of the analyte, a particular temperature and a particularHct. From the measurement points of the series of measurements in FIG.11, it is possible to determine the parameters a and m by an appropriatefit.

With this known calibration curve, it is then possible to generate apiece of correction information in the case of an analyte measurementfrom an individual measurement or multiple measurements of a labelreflectance R_(Label) (i.e., a reflectance caused or influenced by thelabel and its diffusion in the sample). By means of this piece ofcorrection information, it is then possible to correct a measuredanalyte reflectance R_(Analyte) (i.e., a reflectance caused orinfluenced by the detection reaction) and to convert it into a correctedor actual analyte reflectance R_(Analyte)*:

R _(Analyte) *=R _(Analyte) −F(R _(Label))  (6);

R _(Analyte) *=R _(Analyte)−(a+m·R _(Label))  (7).

This corrected analyte reflectance can then, by means of a furthercalibration curve obtained from comparative measurements, be transformedor converted into an analyte concentration such as a glucoseconcentration.

All of the patents, patent applications, patent application publicationsand other publications recited herein are hereby incorporated byreference as if set forth in their entirety.

The present inventive concept has been described in connection with whatare presently considered to be the most practical and preferredembodiments. However, the inventive concept has been presented by way ofillustration and is not intended to be limited to the disclosedembodiments. Accordingly, one of skill in the art will realize that theinventive concept is intended to encompass all modifications andalternative arrangements within the spirit and scope of the inventiveconcept as set forth in the appended claims.

LISTING OF REFERENCE NUMBERS

-   50 Test element-   60 Test support-   100 Test field-   101 Barrier-   102 Test chemistry-   104 Label-   104′ Label-   106 Partition layer-   107 Sample input side-   108 Wetting-   110 Sample/blood-   112 Supernatant-   114 1^(st) region-   116 2^(nd) region-   117 Analyte-   118 Test chemistry layer/reactive layer-   120 Label layer-   122 Pigment layer-   130 1^(st) light source-   140 2^(nd) light source-   150 a Light of the 1^(st) light source-   150 b Light of the 2^(nd) light source-   160 a Reflected light from 1^(st) light source-   160 b Reflected light from 2^(nd) light source-   170 1^(st) detector-   175 1^(st) evaluation unit-   180 2^(nd) detector-   185 2^(nd) evaluation unit-   188 Housing-   189 Opening-   190 Device-   200 Intensity scale-   202 Time scale-   204 1^(st) family of curves-   204 a 0 mg/dl gluc-   204 b 550 mg/dl gluc-   206 2^(nd) family of curves-   206 a 0 mg/dl gluc-   206 b 550 mg/dl gluc-   208 3^(rd) family of curves-   208 a 0 mg/dl gluc-   208 b 550 mg/dl gluc-   210 4^(th) family of curves-   210 a H₂O-   210 b Blood-   212 5^(th) family of curves-   212 a H₂O-   212 b Blood-   214 6^(th) family of curves-   214 a H₂O-   214 b Blood-   216 0.05% erioglaucine-   218 0.1% erioglaucine-   220 0.05% erioglaucine-   222 0.1% erioglaucine-   224 0.05% erioglaucine-   226 0.1% erioglaucine-   228 Family of points having 25% Hct-   230 Family of points having 45% Hct-   232 Family of points having 65% Hct-   234 Measurement point for temperature-   236 Measurement point for Hct-   238 Fitted line

The invention claimed is:
 1. A method of determining at least oneanalyte concentration in a sample having at least one interferingvariable, the method comprising the steps of: detecting at least onediffusion of a diffusable label in the sample or at least part of thesample applied to at least one test element having the diffusable labeland at least one test chemistry to obtain at least one piece ofcorrection information from the detected diffusion of the diffusablelabel, wherein the at least one piece of correction information accountsfor an influence of the at least one interfering variable and accountsfor a general relationship between the at least one interfering variableand diffusion of the diffusable label, and wherein the generalrelationship is obtained from at least one, pre-determined calibrationmeasurement; detecting at least one reaction between the at least oneanalyte and the test chemistry of the at least one test element toobtain an analyte concentration from the at least one reaction of thetest chemistry with the at least one analyte; and correcting the analyteconcentration with the at least one piece of correction information. 2.The method of claim 1, wherein the at least one interfering variable isselected from the group consisting of a temperature of the sample, atemperature of the test element, a proportion of constituents of atleast one substance in the sample, and a combination of at least twothereof.
 3. The method of claim 1, wherein the step of detecting atleast one diffusion of the diffusable label is an electrochemicaldetection, an optical detection or a combination thereof, and whereinthe step of detecting at least one reaction between the at least analyteand the test chemistry is an electrochemical detection, an opticaldetection or a combination thereof.
 4. The method of claim 1, whereinthe test element comprises at least one test field having at least onetest chemistry layer that comprises the at least one test chemistry. 5.The method of claim 4, wherein the at least one test chemistry comprisesat least one enzyme.
 6. The method of claim 5, wherein the at least oneenzyme is selected from the group consisting of glucose dehydrogenaseand glucose oxidase.
 7. The method of claim 1, wherein the diffusablelabel is contained in at least one first region of the test element, andwherein the at least one test chemistry is completely or partlycontained in the at least one first region.
 8. The method of claim 1,wherein the diffusable label is arranged completely or partly separatelyfrom the at least one test chemistry.
 9. The method of claim 1, whereinthe diffusable label does not react with one or both of the analyte orthe test chemistry, and wherein a diffusion rate of the diffusable labelis substantially independent of the analyte concentration.
 10. Themethod of claim 1, wherein the diffusable label absorbs light within awavelength range from about 300 nm to about 800 nm.
 11. The method ofclaim 1, wherein the diffusable label is a dye.
 12. The method of claim11, wherein the dye comprises at least one optically detectable dye. 13.The method of claim 12, wherein the at least one optically detectabledye is hydrophilic or water-soluble.
 14. The method of claim 11, whereinthe dye is selected from the group consisting of cyanine dyes, azo dyes,sulfone dyes, and a combination of at least two thereof.
 15. The methodof claim 1, wherein the diffusable label is selected from the groupconsisting of erioglaucine, indigo carmine, hydroxynaphthol blue,1,1-diethyl-4,4-carbocyanine iodide, amaranth, and a combination of atleast two thereof.
 16. The method of claim 15, wherein the diffusablelabel is erioglaucine or hydroxynaphthol blue.
 17. The method of claim1, wherein the diffusable label and at least one further diffusablelabel different from the first diffusable label are used, wherein adiffusion rate of the first diffusable label is influenced by at leastone first property of the sample, and wherein a diffusion rate of the atleast one further diffusable label is influenced by at least one furtherproperty of the sample different from the first property.
 18. The methodof claim 1, wherein the reaction of the at least one test chemistry withthe analyte is detected by means of at least one first detector, andwherein diffusion of the diffusable label is detected by means of atleast one further detector.
 19. A test element for use in the method ofclaim 1, the test element comprising: at least one test chemistry on atest support of the test element, wherein the at least one testchemistry is configured to carry out at least one detectable reactionwith an analyte, wherein the test chemistry is completely or partlycontained in at least one first region of the test support of the testelement; and at least one diffusable label in the at least one firstregion of the test element, wherein the diffusable label is configuredto diffuse at least partly from the at least one first region of thetest element into at least one second region of the test element. 20.The test element of claim 19, wherein the at least one test chemistrycomprises an enzyme selected from the group consisting of glucosedehydrogenase and glucose oxidase, and wherein the at least onediffusable label is selected from the group consisting of erioglaucine,indigo carmine, hydroxynaphthol blue, 1,1-diethyl-4,4-carbocyanineiodide, amaranth, and a combination of at least two thereof.